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Full text of "Characterization of the rabbit renal H,K-ATPases"

CHARACTERIZATION OF THE RABBIT RENAL H,K-ATPASES 



by 
W GRADY CAMPBELL 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE 

OF DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1998 



This thesis is dedicated to my father, William Maxwell Campbell, and my mother Clara 
Hicks Campbell. 



ACKNOWLEDGMENTS 

I would like to acknowledge the contributions of a number of people who helped me 
in this work. First I would like to thank my committee, the chair Dr. Brian Cain, Dr. 
Charles Wingo, Dr. Susan Frost, Dr. Harry Nick, and Dr. Michael Kilberg. Also Dr. David 
Weiner was very generous to share his lab and microscopes. Dr. Jill Verlander directed a 
very interesting line of research to answer some questions that arose out of our own 
research. 

I would also like to thank my fellow lab members for putting up with me all this time. 
Thanks go to Kim McCormick, Philip Hartzog, Abbe Stack, Jim Gordon, James Gardner, 
Paul Sorgen, Tammy Caviston, and Regina Perry. James, Paul, Tammy, Regina, and I 
shared many years together as a particularly cohesive lab family. Also I want to thank my 
labmates in my lab home away from lab home, Jeanette Lynch, Amy Frank, and Robin 
Moudy. They really went out of their way for me, and it was a pleasure working with 
them. Mary Handlogten was also especially helpful in getting me started in protein work. I 
want to acknowledge the contribution of the Yang lab, including Mike Litt, Chien Chen, 
and Sue Lee. Having an adjacent lab with open doors and shared space between them has 
turned out to be a great design, there has been a high level of camaraderie and exchange of 
ideas between the labs. 



in 



I also appreciate the support and encouragement of my family, my sister Diane, her 
children Emory and Lisacole, my brother-in-law Ken, and the newest addition to our 
family Samma. The companionship and support of Ruth has helped me throughout my 
time here. And most of all, I appreciate the enthusiastic support of my father and late 
mother during my years here. 



IV 



TABLE OF CONTENTS 

ACKNOWLEDGMENTS iii 

LIST OF TABLES vii 

LIST OF FIGURES viii 

ABBREVIATIONS x 

ABSTRACT xiii 

BACKGROUND AND SIGNIFICANCE | 

Overview 

H,K-ATPase in the Kidney 4 

H,K-ATPase Structure and Function 17 

Renal Tissue Culture Cells 23 

Summary 26 

MATERIALS AND METHODS 27 

Molecular Biology 27 

Biochemistry ->g 

Fluorescence Microscopy 40 

FLK-ATPASE (3 SUBUNITS IN THE RABBIT RENAL MEDULLARY 

COLLECTING DUCT 45 

Introduction 45 

Renal Medulla HKp mRNA Variant 46 

Discussion 4 o 

H,K-ATPASE a SUBUNITS IN THE RABBIT RENAL CORTICAL 

COLLECTING DUCT 52 

Introduction S? 

Multiple H,K-ATPase a Subunits in the Kidney 53 

Alternative Splicing of H,K-ATPase a Subunits in the Kidney .... 69 

Expression of H,K-ATPase a Subunits in the Kidney 72 

Discussion .... _, 

/o 



H,K-ATPASE ACTIVITY IN A RABBIT KIDNEY CORTICAL 

COLLECTING DUCT CELL LINE 81 

Introduction 81 

Detection of H,K-ATPase in RCCT-28A Cells 82 

Discussion 100 

PERSPECTIVE AND FUTURE DIRECTIONS 106 

Multiplicity of H,K-ATPase Isoforms in the Kidney 106 

Cell Type Specificity of H,K-ATPase in the Kidney 112 

Future studies 113 

REFERENCES 117 

BIOGRAPHICAL SKETCH 129 






VI 



LIST OF TABLES 

Table page 

2-1 . PCR primer pairs 32 

2-2. Solutions for determination of pH, 41 



VII 



LIST OF FIGURES 

Figure page 

1-1, Schematic diagram of H,K-ATPase 18 

3-1. Northern analysis showing presence of HKP mRNA in renal cortex, renal 

medulla, and stomach 47 

3-2. 3' and 5' RACE reactions to amplify HKp cDNAs using rabbit renal 

medulla as template 48 

3-3. HKp and p' subunit mRNAs 50 

4-1. Design of degenerate primers for RT-PCR of novel P-type ATPases 56 

4-2. RT-PCR product amplified from rabbit renal cortex RNA using degenerate 

primers 57 

4-3. BLAST search using sequence of 419 bp fragment of HKa 2 58 

4-4. Cloning of HKa 2a and HKa :c cDNAs 59 

4-5. GenBank accession records for rabbit HKa 2 sequences 61 

4-6. Northern analysis showing presence of HKa 2a in distal colon and renal 

cortex 67 

4-7. Distance analysis of selected HKa and NaKa subunit coding 70 

4-8. Rabbit HKa 2 gene sequence at the 5' end 71 

4-9. Western analysis showing presence of HKa 2a and HKa 2c protein in renal 

cortex 73 

4-10. Immunohistochemistry by Dr. Jill Verlander and Ms. Robin Moudy 75 

5-1. Southern blots of H,K-ATPase subunit mRNA in RCCT-28A cells 84 

viii 



5-2. HKp and HKa, subunit mRNA in RCCT-28A cells 86 

5-3. HKa 2 subunit mRNA in RCCT-28A cells 88 

5-4. Western analysis showing presence of HKa 2c protein in RCCT-28A cells 90 

5-5. pH, recovery from an acid load by RCCT-28A cells in the absence of Na + .... 96 

5-6. pH, recovery from an acid load by RCCT-28A cells in the presence of EIPA .. 98 

5-7. Summary of the rates of pH ; recovery from an acid load by RCCT-28A cells .102 



IX 



ABBREVIATIONS 



ATP 

BCECF 

BCECF-AM 

bp 

DEPC 

DNA 

EDTA 

EIPA 

FBS 

FITC 

HEPES 

HKa 

HKa, 

HKa 2a 

HKa 2b 

HKa 2c 

HKa 3 



adenosine triphosphate 

2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein 

acetoxymethyl ester of BCECF 

base pairs 

diethylpyrocarbonate 

deoxyribonucleic acid 

ethylene diamine tetraacetic acid 

ethylisopropylamiloride 

fetal bovine serum 

fluoroisothiocyanate 

N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid 

H,K-ATPase a (catalytic) subunit 

H,K-ATPase a, subunit 

H,K-ATPase a 2a subunit 

H,K-ATPase a 2b subunit 

H,K-ATPase oc 2 , subunit 

H,K-ATPase a 3 subunit 



HKou H,K-ATPase ou subunit 

HKp H,K-ATPase p subunit 

HKp' H,K-ATPase p' subunit 

hr hour 

kb thousand base pairs 

kDa thousand Daltons 

M molar 

mEq milliequivalents 

min minute 

MW molecular weight 

NaKa Na,K-ATPase a (catalytic) subunit 

NaKoti Na,K-ATPase a, subunit 

NaKa 2 Na,K-ATPase a 2 subunit 

NaKa 3 Na,K-ATPase a 3 subunit 

NaKou Na,K-ATPase a 4 subunit 

NaKp, Na,K-ATPase p, subunit 

PAGE polyacrylamide gel electrophoresis 

PCR polymerase chain reaction 

pH; intracellular pH 

PMSF phenylmethylsulfonyl fluoride 

pS picoSiemen 

PACE Rapid Amplification of cDNA Ends 

\i 



RNA ribonucleic acid 

RT reverse transcription 

RT-PCR reverse transcriptase-polymerase chain reaction 

SDS sodium dodecyl sulfate 

sec second 

Tris tris[hydroxymethyl]aminomethane 

UTR untranslated region 



XII 



Abstract of Dissertation Presented to the Graduate School 
of the University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 

CHARACTERIZATION OF THE RABBIT RENAL H,K-ATPASES 

By 

W. Grady Campbell 
August 1998 

Chairperson: Dr. Brian D. Cain 

Major Department: Biochemistry and Molecular Biology 

H,K-ATPases are located on the apical membrane of epithelial cells lining the 
collecting duct in the kidney. ATP hydrolysis drives luminal acidification and potassium 
reabsorption by the enzyme. H,K-ATPases consist of two subunits. The catalytic sites are 
located on the a subunits, and the (3 subunits play a role in intracellular trafficking. The 
goal of this work was to elucidate the molecular identity of H,K-ATPase a and p subunits 
in kidney. Northern analysis demonstrated two H,K-ATPase p subunit species in rabbit 
renal medulla; only the smaller of these was present in renal cortex and gastric tissues. A 
search for H,K-ATPase a subunit isoforms in rabbit renal cortex was conducted using 
reverse transcriptase-polymerase chain reaction with degenerate primers. A full-length 
cDNA of the H,K-ATPase a 2 subunit was obtained. Two 5' ends of this transcript were 
observed by 5' Rapid Amplification of cDNA Ends. One (HKa 2a ) had high homology to 

xiii 



previously known H,K-ATPases, and the second was a novel variant (a 2c ). Phylogenetic 
analysis showed that the rabbit a 2a subunit clusters near H,K-ATPases from human axillary 
skin and rat distal colon, but is more distant from gastric H,K-ATPase and 
Na,K-ATPases. Gene sequence analysis showed that the first HKoc 2t exon was located 
within the first HKa 2a intron. Western analysis using antipeptide polyclonal antibodies 
demonstrated the expression of HKcci, HKa 2a and HKa 2c subunits in rabbit renal cortex. 
The mRNAs encoding the HKp, HKa,, HKa 2a , and HKa 2c subunits were detected in the 
rabbit cortical collecting tubule intercalated cell line RCCT-28A. These results indicated 
that acid-secreting intercalated collecting duct cells were a cell type containing 
H,K-ATPase, and these cells appear capable of expressing multiple H,K-ATPase isoforms. 



xiv 



BACKGROUND AND SIGNIFICANCE 



Overview 



H,K-ATPase was first identified in the stomach, where it is expressed at an extremely 
high level to mediate the extrusion of acid that aids digestion. A great deal has been 
learned about regulation of H,K-ATPase in the stomach, enough to feel as though its 
regulation is understood in a meaningful way. At the molecular level, gastric-specific 
transcription factors have been found that may be responsible for the marked 
tissue-specific regulation of expression of H,K-ATPase (Maeda et al, 1991; Oshiman et 
al, 1991; Tamura et al, 1992). The principal means of induction of gastric H,K-ATPase 
from its basal state is by unwinding a tightly coiled tubular network, exposing pumps 
localized in the network and thereby increasing the size of the secretory surface (Pettit et 
al, 1995). This morphological transformation is triggered by various stimuli including 
activation of stretch receptors in the stomach by entering food, the sight of food, or even 
the thought of food (reviewed by Wolfe and Soil, 1988; Hersey and Sachs, 1995). Thus, 
there is a framework of understanding of the regulation of H,K-ATPase in the stomach at 
the molecular and cellular levels, and the root stimuli that result in the H,K-ATPase 
response are known. 

H,K-ATPase is expressed at much lower level in the kidney, but its role in the kidney 
is even more critical to life than its role in the stomach. The kidney plays the major role in 

I 



2 
K + homeostasis, maintaining extracellular (including plasma) K' concentration within the 

relatively narrow normal range of 3.8 to 5.0 mEq/L (Merck, 1992; Guyton and Hall, 

1997) despite a wide variation in K + intake. If plasma K + levels depart significantly from 

these values, then cardiac arrhythmias lead to life-threatening conditions. 

K + transport in the kidney is also important to blood pressure regulation. It has been 
observed that urinary K + levels and urinary Na7K + ratio are related to systolic and 
diastolic blood pressures (INTERSALT, 1988; Whelton, 1993). Excretion rates show a 
higher degree of correlation with elevated blood pressure than serum levels, implicating 
the kidney as an important organ in regulating the effect of K + on blood pressure. Dietary 
K + supplementation has been shown to lower blood pressure since early this century 
(Ambard and Beaujard, 1904; Addison, 1928). 

The substantial role of renal ion transport in blood pressure regulation is emphasized 
by studies conducted by Lifton (1996), who determined the molecular bases underlying 
certain types of monogenic inherited extreme hypo- and hypertension disorders. Each of 
the genetic defects found affected renal ion transport. Such well-defined inherited diseases 
of blood pressure regulation are not common. In 95% of people experiencing hypertension 
their diagnosis is essential hypertension, the root cause being essentially unknown. To 
understand this majority of hypertensive disorders, a more complete understanding of 
renal ion transport is needed. Because of the role of H,K-ATPase in the final regulation of 
K + excretion in the kidney, understanding its contribution to renal ion transport could well 
be important in understanding the problem of essential hypertension. 

In order to understand the role of H,K-ATPase in the kidney, the exact H,K-ATPase 
pumps of the kidney must be characterized at a molecular level. This characterization must 



3 
be carried out to a high level of detail, to explore variations in the ion pumps conferred by 

differences in changes in transcriptional start or adenylation sites, or by alternative 
splicing. With the identity of the H,K-ATPase subunits known at a molecular level, 
various reagents can then be developed to characterize H,K-ATPase regulation. These 
include cDNA probes, antibodies, and activity assays. With these tools available, it will be 
possible to develop a more complete framework of understanding of renal H,K-ATPase. 

The studies in this dissertation contribute to our knowledge of the H,K-ATPase 
subunits at a molecular level. At the time these studies began, it was not known what 
H,K-ATPase subunits were responsible for the active H + and K + exchange that had been 
observed in renal collecting duct. Further evidence is presented that all the currently 
known H,K-ATPase subunits are present in kidney, and that the primary cell type in which 
all are expressed is the collecting duct acid-secreting intercalated cell. When these studies 
were begun, there was no known alternative splicing of P-type ATPases. Here alternative 
splicing of an HKa subunit mRNA is described, and expression of a protein product is 
shown in kidney. In addition, a variant renal medulla HK0 transcript is described that has 
tissue-specific expression even within the kidney. In summary, this work has generated a 
new appreciation for the complexity of H,K-ATPase molecules that underlie renal 
collecting duct H,K-ATPase activity. 



H,K- ATPase in the Kidney 
Transport activity 

The kidney is the principal organ responsible for potassium homeostasis and plays a 
major role in maintenance of the acid-base balance of the body. The renal collecting duct 
(CD) is the primary site of regulation of the excretion of potassium and the acidification of 
urine. A number of studies implicate an apical H,K-ATPase as an important mediator of 
these functions. The enzyme (pump) actively transports H + into the lumen of the nephron 
in a nonelectrogenic exchange for IC (for review, see Wingo and Cain, 1993, Wingo and 
Smolka, 1995). 

A K + activated ATPase was observed in frog gastric microsomes by Ganser and Forte 
(1973). Lee et al. (1974) found that gastric microsomes isolated from dog mucosa were 
able to accumulate H + in the presence of ATP and IC. These studies were the initial 
observations of the now well-known gastric H,K-ATPase. The designation of 
H,K- ATPase was given the enzyme by Sachs et al. (1976). 

Gustin and Goodman (198 1) isolated apical brush-border membrane of the rabbit 
descending colon by isolation of epithelial cells, homogenization, and centrifugation on a 
Percoll gradient. They found a membrane-bound, K-activated ATPase, which had a 
Kac,=2xlO^M for K\ was competitively inhibited by Na + , but had no activation by Na + . It 
was vanadate sensitive, but oligomycin and ouabain (ImM) insensitive. This represented 
the first observations of enzymatic activity for the colonic H,K- ATPase. 

Smolka and Sachs (in Sachs et al., 1982), employing monoclonal antibodies, detected 
a protein at least similar to the gastric proton-potassium-translocating ATPase protein in 



5 
renal distal tubule and colon. Sachs el al. (1982) advanced the idea that this protein might 

be involved in the K + reabsorption or the acidification known to take place in kidney or 
colon. 

By quantitating the hydrolysis of [y- 32 P]ATP, Doucet and Marsy (1987) observed a 
K + -stimulated ATPase activity in rabbit kidney. The level of activity was related to the 
density of intercalated cells in microdissected segments of rabbit connecting segment 
(highest activity), cortical collecting duct (intermediate), and outer medullary collecting 
duct (lowest), and not detectable in any other nephron segments. The ATPase affinity for 
K + was high (K m =0.2-0.4mM). The pharmacological properties of the renal H,K-ATPase 
was examined as a preliminary indication of the pumps present in the kidney. Omeprazole 
is an inhibitor of the gastric isoform of H,K-ATPase, vanadate inhibits all P-type ATPases, 
and ouabain is a Na,K-ATPase inhibitor that also inhibits non-gastric H,K-ATPase 
isoforms. The renal ATPase was inhibited by omeprazole and vanadate, but not by 
ouabain. They also observed a K + -ATPase activity with potassium restriction in rat renal 
outer medullary collecting duct that roughly doubled, changing little after 0.5 weeks 
low-K + diet. In cortical collecting duct activity doubled, rising steadily during a five week 
low-K + diet (Doucet and Marsy, 1987). 

Wingo (1987) did a more complete study of dietary K f influence on K + transport in the 
kidney. He first established that rabbits on a K + -replete diet, or on a K-depleted diet, or 
on a K + -deplete Na + -supplemented diet all consumed similar quantities. Wingo (1987) 
used restricted diets containing 0.55% K\ marginally less than the 0.6% generally thought 
to be required for normal rabbit growth. In time-course studies he found that after an 
initial two-week period of K + -replete meals, 72 hr was sufficient for the response to 



6 
equilibrate as judged by urinary Na* and K" excretion. Muscular K" levels had not altered 

significantly in that time, showing that renal response preceded effects deleterious to the 

animal. Serum K + levels were slightly higher in K' -replete animals, and the same in 

K + -depleted Na + -supplemented animals compared to K'-depleted animals. The sodium 

supplementation evidently maintained the IC serum level. Serum aldosterone was found to 

be 2.88±.57 ng/dl in K + -depleted Na"-supplemented rabbits, 15.6±5.3 for K + -depleted 

rabbits, and 44.7±14.0 for potassium-replete animals, and thus any changes in transport 

observed cannot be correlated with aldosterone level. 

In his experiments, Wingo (1987) found that perfused collecting ducts from outer 
medullary inner stripe had similar rates of fluid reabsorption, gauged by 3 H-inulin flux, and 
similar transepithelial voltages. K' reabsorption, however, roughly tripled among rabbits 
on K + -restricted diets, measured as a flux of 42 K. K f -replete rabbits had 
5.9±0.8%,K + -depleted rabbits had 17.2±2.0%, and K-depleted Na + -supplemented rabbits 
had 20.8±4.2% reabsorption. The data were not significantly different in either of the two 
K + -restricted cases. He concluded that K* reabsorption in the medullary collecting tubule 
is comparable to K + secretion in the cortical segments at normal fluid flux rates, and could 
have important contributions to the amount of K H excreted in urine. In a later report by 
Wingo (1989), the name H,K-ATPase was first explicitly given to the K + -stimulated 
ATPase that had been studied by others in the kidney. 

Utilizing a fluorometric microassay in which ATP hydrolysis is coupled to the 
oxidation of NADH, Garg and Narang (1989) detected the presence of a K + -dependent, 
ouabain-insensitive ATPase activity in rabbit kidney that was also omeprazole-, 
SCH28080-, and vanadate-sensitive. Sch-28080, like omeprazole, is a potent gastric 



7 
H,K-ATPase inhibitor. Earlier work by this group (Garg and Narang, 1988) noted H + 

secretion coupled to an ATPase that was not NEM-inhibited, and could not account for 

the vacuolar IT -ATPase they were studying. Significant K' -ATPase activity was seen in 

microdissected connecting segment (17.0±3.3 pmol min' 1 mm" 1 ), cortical collecting duct 

(6.6±0.7), and outer medullary collecting duct (8.8±1.7), but not in proximal straight 

tubule, convoluted tubules, nor the thick ascending limbs. They found the K + -ATPase 

activity to be affected by diet. Activity was found in the connecting segment (13.0±4.0 

pmol min" 1 mm"'), cortical collecting duct (10. 1 ±3.0), and outer medullary collecting duct 

(10.8±2.2) in animals on a low K' diet. Cortical collecting duct activity varied with pH; 

optimal pH of 7.4 gave activity of 10 pmol min" 1 mm 1 , falling at lower pH to 3 at pH of 

7.0, and falling at higher pH to 5 at pH 7.8. Thus, a high K r diet completely suppressed 

the K + -ATPase activity, while rabbits fed a normal diet had similar activity to the low K + 

fed rabbits of the second study. 

Studies by Cheval and co-workers (Cheval et a/., 1991) examined the S6 Rb flux ( 86 Rb is 

a K + analog used as a radioactive tracer for transporter studies) and K-ATPase activities. 

These activities were blocked by Sch-28080 in cortical and medullary collecting duct. 

Activities they attributed to Na,K-ATPase and H-ATPase were unaffected by Sch-28080. 

In this study, ouabain was used at a concentration of 2.5 mM to inhibit Na,K-ATPase. 

Although there are inconsistencies in the exact concentration at which ouabain inhibits 

H,K-ATPases containing the HKa 2 subunit, all expression studies carried out to date 

(Modyanove/a/., 1995; Codina et a/., 1996; Cougnon etal., 1996; Grishin eta/., 1996) 

except for one (Lee et «/., 1995) indicate that 2.5 mM ouabain is sufficient to block the 

HKa 2 enzyme. Lee et at. (1995) detected no ouabain sensitivity at 1 mM, and it is not 



8 
known what effect 2.5 inM would have had in their system. In this work by Cheval et al. 

(1991) the activities of Na,K-ATPase and an HKa 2 H,K-ATPase would be 

indistinguishable. 

These early studies do not distinguish between different isofonns of H,K-ATPase that 

may together account for the H,K-ATPase activity observed in the kidney. Given 

historical perspective, this makes sense because at the time these studies were conducted, 

only HKcti and HKP subunit isoforms were known, and the ouabain-sensitive 

H,K-ATPase was not yet known. Later studies (Younes-lbrahim et al., 1995; 

Buffin-Meyer et al, 1997) addressed the issue of multiple isoforms of H,K-ATPase in the 

kidney and their distribution along the nephron and collecting duct. These investigators 

found three distinct H,K-ATPase activities. Two were found in the collecting duct; one of 

these was sensitive to Sch-28080; the other was insensitive. The third was found in 

proximal tubules and thick ascending limbs, and was inhibited by Sch-28080. In normal 

rats, the sole H,K-ATPase activity present in collecting duct was the Sch-28080-sensitive 

activity. In K + -depleted rats, the overall H,K-ATPase activity in collecting duct increased, 

while the activity in proximal tubule decreased. The increase was abolished by ouabain, 

but not by Sch-28080, implying that the increase is due to an H,K-ATPase isoform that is 

pharmacologically dissimilar to HKa,. The proximal tubule/thick ascending limb 

H,K-ATPase may have a basolateral polarity, rather than the apical localization of the 

collecting duct H,K-ATPase. If the H,K-ATPase role in proximal tubule and thick 

ascending limb involves K + homeostasis, then its down regulation by dietary K + depletion 

would imply a basolateral location. Together, these results argue for three different 

H,K-ATPase isoforms in kidney. One was constitutively expressed in the collecting duct, 



and was Sch-28080-sensitive and ouabain-insensitive, like the HKcti isoform of 
H,K-ATPase. The second was presumably located basolaterally in proximal tubule and 
thick ascending limb, reduced by low K', and was ouabain-sensitive. The third was located 
apically in the collecting duct, stimulated by low K\ and Sch-28080-insensitive but 
ouabain-sensitive. 

Earlier studies naturally concentrated on establishing the existence of H,K-ATPase in 
the kidney. The early studies demonstrated H,K-ATPase activity in connecting segment, 
cortical collecting duct, and outer medullary collecting duct. Although measurements were 
made for H,K-ATPase activity in other nephron segments, no H,K-ATPase activity was 
found outside the connecting segment and collecting duct. More recently, proximal tubule 
and thick ascending limb have been added as regions having H,K-ATPase activity. These 
studies also began to address the differences among H,K-ATPases that are present in the 
kidney, defining the characteristics of the H,K-ATPase activities and their localization. 
The identity of the H,K-ATPase molecules responsible for the two collecting duct 
activities have been found. The identity of the H,K-ATPase molecule responsible for the 
activity in the more proximal nephron is not yet known. 

H.K-ATPase subunit isoforms in the kidney 

Until the last five years, the presence of H.K-ATPase in the kidney has been defined 
only on the basis of activity. We have now begun to determine the molecular identities of 
the pumps responsible for this activity. And only very recently it has become appreciated 
that the complexity of H,K-ATPase expression also includes alternative splicing of HKoc 
and HKfS isoforms. 



10 
An H,K-ATPase has long been known to acidify the lumen of the stomach. This pump 

is a member of the P-type ATPase family, which shares similarity of sequence, structure 

and mechanism. The gastric H,K- ATPase is comprised of a catalytic a, subunit and a P 

subunit; the active form of the enzyme is an (a(3) 2 oligomer (for review see Hershey and 

Sachs, 1995; Van Driel and Callaghan, 1995). Full-length cDNAs for rabbit HKa, 

(Bamberg et ai, 1992) and HKp (Reuben et a/., 1990) have been cloned and sequenced. 

RT-PCR followed by sequencing of the amplified products has been used to demonstrate 

that mRNA (Ahn and Kone, 1995) and protein (Callaghan et a/., 1995) for HKa, and that 

mRNA and protein for HKp (Callaghan et a/., 1995) isoforms are present in kidney. This 

author was included in the latter study. 

The non-gastric H,K-ATPase a subunit isoforms (HKa 2 ) have been cloned from 

several tissues. A partial cDNA for an HKa 2 isoform was obtained from human axilla skin, 

and mRNA observed in brain and kidney (Modyanov el ai, 1991). A full-length cDNA 

was found subsequently (Grishin et ai., 1 994). An HKot 2 cDNA was cloned from rat distal 

colon by Crowson and Shull (1992); the corresponding amino acid sequence has 86% 

amino acid identity to the cDNA derived from human skin. This rat HKoc 2 mRNA was 

detected in kidney, uterus, and heart using two separate cDNA probes from HKa 2 3' UTR 

and C-terminal transmembrane domains. The 3' UTR derived probe detected HKa 2 in 

forestomach as well. These human and rat cDNAs were shown to encode H7K + exchange 

activity when expressed in Xenopus laevis oocytes (Modyanov et ai., 1995; Cougnon et 

al, 1996), although recent work questions the stoichiometry of the exchange in the human 

isoform (Grishin et ai, 1996). In two of these studies rabbit HKp subunits were 



11 

cotransfected (Modyanov et al, 1995; Grishin et al, 1996), and in the other Bufo marinus 
HKp subunits were cotransfected (Cougnon et al, 1996). A partial HKa 2 cDNA was 
cloned from a rabbit cortical CD (CCD) library having 84% amino acid identity to the 
human HKcc 2 , and mRNA was detected in CCD and colon (Fejes-Toth et al, 1995). 
Watanabe et al. (1992) cloned and sequenced a similar cDNA from distal colon of guinea 
pig. The degree of identity at the amino acid level among human, rat, guinea pig, and 
rabbit HKa 2 clones is less than the amino acid identity of HKcti (>97%) between the three 
species, but much greater than the typical amino acid identity between HKa, and other 
P-type ATPases (<64%). Therefore, although controversy on this point exists, this author 
considers these to be orthologous and refers to them collectively as HKa 2 . As part of this 
dissertation, further evidence will be presented that the HKa 2 genes are indeed orthologs. 
A related cDNA (75% identity at the amino acid level) was cloned from Bufo marinus 
bladder, an analog of mammalian collecting duct (Jaisser et al., 1993). While mRNA was 
detected in toad bladder, none was observed in either stomach or colon. The evolutionary 
distance between toad and mammal coupled with the tissue distribution dissimilarity of the 
toad HKa make it difficult to evaluate the relationship between the toad and mammalian 
isoforms. In addition to uncertainty in the number of H,K-ATPase isoforms in the kidney, 
it has been recently been discovered that there are two alternatively spliced transcripts of 
the rat HKct 2 in kidney (Kone, 1996, Higham and Kone, 1998). Thus, several 
H,K-ATPase a subunit isoforms have been reported in kidney, but it is at present 
uncertain whether these account for all the renal H,K-ATPases. Also uncertain is their 
relative contributions to renal H,K-ATPase activity. 



12 
Experiments have been conducted in attempts to find more members of the gene 

family that includes the a subunits of Na,K-ATPases and H,K-ATPases. Shull and Lingrel 
(1987) probed a human genomic library at low stringency with probes made from 
Na,K-ATPase sheep a u rat cti, rat a 2 , and rat a 3 . Five different sequences were obtained, 
with two to ten clones representing each sequence. Three of these were known to be 
human NaKa,, NaKa 2 , and HKa, genes. The fourth was later identified as HKa 2 
(Modyanov et al., 1991). The fifth, which is physically linked to the NaKa 2 , was later 
identified as NaKou by Shamraj and Lingrel (1994). In a similar experiment, Sverdlov et 
al, (1987) screened a human genomic library with a probe made from porcine kidney 
NaKcti. The probe was constructed to contain the well-conserved region surrounding the 
active site aspartate residue. They obtained five distinct clones. They were recognized as 
the three known NaKa subunits and the two known HKa subunits. In sum, the results of 
these two experiments imply that all members of the gene family of X,K-ATPases are 
known. 

At present, there are no known specific HK(3 isoforms in addition to the one originally 
discovered in gastric tissues. However, it is thought that the NaKp, subunit isoform is the 
partner to HKa 2 in active H,K-ATPase pumps in colon and kidney (DuBose et al, 1998; 
Kraut et al, 1998). These observations are the first suggesting that an individual P-type 
ATPase P subunit has more than one primary P-type ATPase a subunit partner in vivo. 
This was the determination reached by two separate groups using different antibodies to 
specifically immunoprecipitate ap pairs, lending strength to their independent and identical 
conclusions. However, it is possible that during tissue processing some of the a/p pairs 



13 
may mix partners, leading to an erroneous conclusion. Pairs that are not thought to 

associate in vivo have been seen in expression systems to give rise to functional activity 
(Horisberger el al., 1991; Codina el al, 1996), showing that the in vivo P-type ATPase 
pairs are not exclusive when expressed in expression systems. Expressed in their proper 
cell types, there may be compartmentation that controls HKa and HKP selectivity. This 
could segregate the subunits from their incorrect partners on the basis of translation of the 
various H,K-ATPase and Na,K-ATPase isoforms at different times or in different 
locations. 

Alternative transcriptional start sites for HKP in the stomach were detected by 
Newman and Shull (1991). The primer extension method used by Newman and Shull 
(1991) would not have necessarily differentiated between alternative transcriptional start 
sites and alternative splicing. Primer extension would only give information about the 
distance from the primer to the beginning of any or all of the transcripts that contain the 
primer site. So it is possible that they were detecting alternative splicing as well as 
alternate transcription start sites. Thus far, however, there have been no reports of 
multiple HKP subunit transcripts in any tissue seen by RNA analysis techniques such as 
northern analysis or ribonuclease protection assay. In present work, I present evidence 
that the transcriptional start site ofHKp subunit in the medulla has slight differences 
relative to stomach, and that in renal medulla there are two transcripts expressed at 
comparable level, one of which appears to be the product of alternative splicing. 

In summary, the first H,K-ATPase subunit detected in kidney was the non-gastric 
isoform HKa : , shown by RT-PCR to be present in human kidney (Modyanov et al., 
1991). Shortly before this author joined his lab, Dr. Brian Cain found that the rabbit P 



14 
subunit isoform of stomach was present in kidney (Callaghan et a/., 1995; 

Campbell-Thompson et a/., 1995). After the work described here was begun, work by 
other investigators (Ahn and Kone, 1995) established that the catalytic subunit isoform 
found in stomach, HKcci, is also found in kidney. HKa, protein was shown by immunoblot 
in rabbit kidney (Callaghan et al:, 1995). To further complicate the picture, an 
alternatively spliced HKa 2 (dubbed HKa :b , with the canonical HKct : being renamed 
HKa 2a ) was discovered in kidney (Kone and Higham, 1998). A search for the P subunit 
partner to HKa 2a established that NaK(3, couples to HKa2 a in vivo (Dubose et al., 1998; 
Kraut etal, 1998). Presumably, HKa, pairs with HK(3 in kidney as it does in stomach 
(Hall etal, 1991; Shin and Sachs, 1994, Mathews et al.; 1995). All the known 
H,K-ATPase subunit isoforms are known in kidney. HKa,/HKP and HKa 2 /NaKp, 
H,K-ATPases contribute to the activities observed in the collecting duct. Experiments 
have been done to find other candidate H,K-ATPase genes, and to date none have been 
detected. So far no molecular identity can be associated with the H,K-ATPase activity in 
the proximal tubule and thick ascending limb. 

Deficiencies of H.K-ATPase 

There are two lines of evidence to indicate that H,K-ATPases are critical to 
maintenance of K + homeostasis, and that disturbances in this balance are a serious health 
problem. First, transgenic mice with deranged H,K-ATPase function show substantial 
disturbances of K + balance (Meneton et al, 1998). Second, an environmentally high level 






15 
of vanadium in northeast Thailand effectively inhibits H,K-ATPase function among 

humans and water buffalo living in the area (Dafnis et a/., 1993). 

Experiments have been carried out to show the critical nature of H,K-ATPase 
expression and its relevance in the kidney (Meneton et a/., 1998). Meneton showed that 
transgenic mice homozygous with respect to an HKa 2 subunit deletion were normal when 
fed diets that had normal levels of K" (1% K + ). However, when fed K + -free diets 
(<0.004% K + ) these mice experienced loss in body weight, plasma K\ and muscle K + . 
HKa 2 is not the major mechanism of K conservation in the kidney; the urinary K + 
excretion rate in both wild-type and HKa-deficient mice declined 100-fold compared to 
HKa 2 -deficient mice on normal diets. However, mean urinary K + excretion per day was 
consistently higher (typically 120% of wild-type) following a week of K + -free feeding. 
This suggests that there may be a role played by renal HKa : in K/ conservation. The role 
played by HKoc 2 in the colon was more clear. Fecal excretion rate increased four-fold in 
HKa 2 -deficient compared to normal mice that had been fed the same K + -free diet, 
indicating an inability to reabsorb IC by the digestive tract. Cardiac arrhythmias were 
observed in some of the HKa : -deficient mice, presumably due to low plasma K + . 

Anecdotal evidence concerning H,K-ATPase activity in humans comes to us from 
Thailand, where environmental vanadium levels are high. Vanadate is a transition state 
inhibitor for all P-type ATPases, and its presence in drinking water and soil in northeast 
Thailand was associated with an epidemic of renal distal tubular acidosis. It is interesting 
that although vanadate would be expected to inhibit Na,K-ATPases and Ca-ATPases, 
along with H,K-ATPase in stomach and distal colon, the predominant symptoms were 



16 
renal. The disease is characterized by an inability to lower urine pH to below 5.5 pH units. 

Affected persons had generalized paralysis, hypokalemia, metabolic acidosis, muscle and 
bone pain, and nocturia (Nilwarangkar el al., 1990). The connection between acid 
secretion in the kidney and stomach had been made, and some patients tested positive for 
gastric hypoacidity (Sitprija et al., 1988). However, the exact cause of the acid secretion 
defect was unknown. As recently as 1990, a genetic predisposition had not been ruled out 
(Nilwarangkar eta/., 1990). Patients are treated by K + and alkali supplements, and some 
deaths occurred with non-compliance. 

It was shown in rats that intraperitoneal injections of vanadate (5 mg/kg) led to 
hypokalemic distal renal tubular acidosis and loss in muscle K + (Dafnis et al, 1993). 
Cortical collecting duct K-ATPase activity that was sensitive to Sch-28080 (200 u.M) 
declined 75% in vanadate-treated rats. Medullary collecting duct activity declined less than 
50% by the same assay. However, it should be noted that this is a sufficiently high 
Sch-28080 concentration to inhibit HKa : enzyme activity according to some investigators 
(Modyanov etal, 1995; Grishin et al., 1996), but other studies have not seen sensitivity 
to Sch-28080 even at that relatively high concentration (Cougnon et al, 1996; Codina et 
al, 1996). Therefore, a vanadate effect on HKa 2 enzyme activity in this study might have 
remained undetected. Na,K-ATPase activity was measured also, and showed a decline in 
vanadate-treated animals as well. This assay also may or may not have detected a 
contribution due to HKa 2 activity, which is expected to be ouabain-sensitive at 
concentrations of between >10 uM (Modyanov et al., 1995) and <1 mM (Codina et al., 
1996) depending on the study. The evidence suggests that vanadate could be a factor in 
the endemic hypokalemic distal renal tubular acidosis in northeast Thailand. More precise 



17 
measurements need to be made to quantitate the effect of Sch-28080 and ouabain on 

HKoc 2 activity. 

In summary, these studies illustrate the essential nature of H,K-ATPases in the renal 
collecting duct (and in distal colon) for the maintenance of K r balance. In the extreme 
case, deranged H,K-ATPase function can result in disease or even death. These studies 
isolating the activity of H,K-ATPase do not address the role H,K-ATPase might play in 
blood pressure regulation. The other more acute affects of impairments of H,K-ATPase 
activity probably obscured the affects on blood pressure regulation. Although in some 
cases a defect in a single gene leads to a dramatic loss in blood pressure control, 
hypertension is most often a multifactorial disease. H,K-ATPase is likely to be one of a 
combination of activities that fail to control blood pressure in the hypertensive patient. 

HK-ATPase Structure and Function 

H,K-ATPase catalytic subunit 

The HKa subunit contains the active sites relating to its catalytic action (Figure 1-1). 
A number of functionally important sites in the HKa have been defined. The location of 
the phosphorylated aspartyl residue of the catalytic intermediate is known and the region 
immediatedly surrounding it is extremely well conserved within the P-type ATPase family 
(Walderhaug eta/., 1985). The location of a residue that binds the fluorescent molecule 
FITC is also known; FITC competes with ATP for binding to H,K-ATPase and therefore 
is thought to be at or near the HKa ATP binding site (Jackson el al., 1983; Farley and 
Faller, 1985). The site of the ^-competitive inhibitor Sch-28080 which acts at an 









18 



extracellular 







ATP ADP+P, 



intracellular 



Figure 1 - 1 . Schematic diagram of H,K- ATPase. Dashed line indicates the 
amino-terminal extension of HKa 2c protein. 



19 
extracellular site (Munson and Sachs, 1988) is known by studies with a photoaffinity 
analog to lie between the first two transmembrane domains (Munson et al., 1991). The 
Na,K-ATPase inhibitor ouabain binds to the corresponding site in that enzyme, but it is 
not known whether this is the binding site conferring ouabain's less sensitive inhibition of 
HKa 2 . The binding site of the medically important inhibitor omeprazole is also known, it 
may bind to any of three extracellular cysteines located in the C-terminal quarter of the 
subunit (Besancon et al., 1993). This inhibitor has achieved some popular acclaim, 
television advertisements may be seen for it under its brand name Prilosec. 

Using hydropathy analysis, the existence of four transmembrane helices in the 
amino-terminal half of HKcti is clear. The number of transmembrane domains of the 
carboxyl-terminal half of HKa is not as apparent from the hydropathy plot, but may be 
placed somewhere between three and five by that analysis. However, the presence of an 
odd number of membrane-spanning regions would be inconsistent with the placement of 
amino- and carboxy-termini in the cytoplasm by antibody reactivity (Smolka et al., 1992; 
Mercier et al, 1993). Also, the localization of the active sites discussed in the preceding 
paragraph, predicts a large intracellular cytosolic loop following the first four 
transmembrane domains. The picture is further complicated because recent results 
(Raussens et al., 1998) imply that some of the transmembrane domains may be comprised 
of P-strand secondary structure. Using limited tryptic digestion followed by fluorescent 
labelling of cysteines, the four transmembrane segments of the amino-terminal half could 
be confirmed (Besancon et al., 1993; Shin et al., 1993). In the carboxyl-terminal half of 
the protein, only three transmembrane domains were observed. The two most 
carboxyl-terminal membrane spans strongly implied by hydropathy analysis were not 



20 
observed by fluorescent labelling despite the presence of multiple cysteines. In vitro 

translation experiments have also been preformed to elucidate H,K-ATPase topology 
(Bamberg and Sachs, 1994). These experiments involve synthesis of putative 
transmembrane segments in the presence and absence of microsomes, then detection of 
glycosylation of the resulting polypeptides by electrophoresis. Positive glycosylation of a 
fusion protein containing glycosylation sites would imply the insertion of a single 
transmembrane domain. When a pair of membrane spanning domains were translated, no 
glycosylation would result. Both single transmembrane domains and pairs of 
transmembranes were tested. In this system, the predicted final two transmembrane 
segments were inserted into the membrane. Taken together these experiments predict a 
secondary structure for HKoci that has amino- and carboxy-termini located intracellularly. 
There are four transmembrane domains in the amino-terminal half of the protein, and 
between four and six transmembrane domains in the carboxyl-terminal half. If 
H,K-ATPase membrane topology is conserved with that of Ca-ATPase, recently imaged 
by cryoelectron microscopy (Zhang el al., 1998), then HKa, subunits probably have ten 
membrane spans. 

The significance of H,K-ATPase enzyme quaternary structure is presently under active 
consideration. Studies suggest that there are specific and stable associations between 
catalytic subunits. Radiation inactivation studies showed the minimum functional unit to be 
an (ap) 2 heterotetramer (Rabon el al., 1988) The electron microscope diffraction pattern 
in images of two-dimensional H,K-ATPase crystals suggested a tetrameric arrangement of 
HKa, subunits (Hebert el al., 1992). The apparent contradiction of the radiation 
inactivation and electron microscopy experiments can be explained. An a/a interaction has 



21 
been shown to be necessary for optimal activity of H,K-ATPase (Morii et al, 1996). 

Dimeric a/a interactions were necessary for H,K-ATPase activity, and tetrameric a/a 

interactions confer higher affinity binding of ATP. When coexpressed in insect cells, 

NaKai, NaKa 2 , and NaKa 3 were found to coimmunoprecipitate (Blanco et a/., 1994). 

Using chimeric constructions combining NaKa, and HKa, alternating regions, it was 

found that the large intracellular loop was necessary for this association (Koster et al., 

1995). Using a yeast two-hybrid system and fusions of Gal4 to a subunit cytosolic loops 

Colonna etctl. (1997) further explored this interaction. In two-hybrid assays, there was no 

apparent direct interaction between pairs of the large cytosolic loop (Figure 1-1). Likewise 

there was no interaction observed between the smaller loop between the second and third 

transmembrane domains. However, the two-hybrid assay gave a positive interaction 

between the smaller and larger loops. The interaction is apparently between the loop 

between transmembrane domains two and three of one member of the a/a pair and the 

larger loop between transmembrane domains four and five of the other. These results 

imply that interactions between H,K-ATPase molecules play a role in their function. There 

may be interactions with other molecules as well, such as those governing cell polarity. 

H,K-ATPase B subunit 

Antibodies to the HKP subunit protein (Chow and Forte, 1993) and reduction of any 
of the three disulfide bonds in the HKfj subunit (Chow el al., 1992) have been observed to 
affect catalytic subunit function. In addition to a role in modulating enzymatic activity, the 
HKp subunit is also thought to participate in shepherding the H,K-ATPase through the 



22 
Golgi and to the plasma membrane (Renaud el a/., 1991). The HK|3 subunit contains a 

tyrosine-based signal required for internalization of the H,K-ATPase pump 

(Courtois-Coutry et a/., 1997) and thus required for proper regulation of pump activity. 

The HK(3 subunit protein contains 7 consensus N-glycosylation sites and all are 

glycosylated (Chow and Forte, 1993). Thus, the HKP subunit is required for H,K-ATPase 

pumps reaching the cell surface and proper function of the enzyme. Its role in 

internalization means that it is required to down-regulate gastric acid secretion. 

Hydropathy analysis predicts a single membrane domain for the HKP subunit protein. 
Interactions between HKa and HKP subunit proteins have been studied by limited tryptic 
digestion. After digestion, detergent solubilization, and lectin binding of the HKp subunit, 
an HKa subunit fragment corresponding to the putative loop between transmembrane 
domains seven and eight was recovered (Shin and Sachs, 1994). The sequence of this 
fragment is well-conserved, implying some important function. Yeast two-hybrid analysis 
has confirmed this area of the HKa subunit protein (Arg-898 to Arg-922) as a region of 
association with the HKp subunit (Melle-Milovanovic el a/., 1998). The yeast two-hybrid 
analysis has also shown that two extracellular domains of the HKp protein to be regions of 
association with the HKa subunit. The HKp subunit amino acids involved are Gln-64 
through Asn-130 (adjacent to the membrane) and Ala- 1 56 to Arg-188. 

The HKp subunit is quite important to H,K-ATPase activity due to its role in 
intracellular trafficking of the holoenzyme. It apparently also affects the conformation of 
the intact complex, because HKp-specific effects modify enzymatic activity. Just as there 
are a/a interactions that are now known, the regions of the subunit important to a/p 






23 
interactions are also becoming known. These may be important in conferring the effects on 

enzyme activity conferred by oligomeric structure. They may also be important in 

controlling the interaction between the various a and P subunit proteins of the 

H,K-ATPases and Na, K-ATPases. 

Renal Tissue Culture Cells 

A number of renal continuous cell lines exist, but for the purpose of studying 
H,K-ATPase relevance to the cortical collecting duct the selection narrows considerably. 
In cortical collecting duct H,K-ATPase activity is relatively high, and based on 
immunohistochemical (Wingo el a/., 1990) and in situ hybridization evidence 
(Campbell-Thompson ei al., 1995; Aim and Kone, 1995). H,K-ATPase is found in 
intercalated cells in cortical collecting duct. There are two cell lines with characteristics of 
these cell types, MDCK cells and RCCT-28A cell. 

The Madin-Darby canine kidney (MDCK) cell line is an epithelial cell line that 
appears to have originated from distal tubule (Herzlinger el al., 1982) or cortical 
collecting tubule (Valentich, 1981), and has properties of intercalated cells (Pfaller etal., 
1989). MDCK cells are aldosterone-responsive (Simmons, 1978), and immunostaining of 
H + /K + ATPase has been observed (Adam Smolka, personal communication). Oberleithner 
etal. (1990) detected an aldosterone-stimulated, omeprazole-inhibited transport activity 
that was blocked by increased apical extracellular [H], or decreased apical extracellular 
[K + ], consistent with the presence of an apical H7K 1 ATPase in MDCK cells. MDCK cells 
are available for purchase from the American Type Culture Collection (ATCC, Rockville, 
MD). Because of the uncertainty of their origin, and because we wanted to take full 



24 
advantage of the knowledge of rabbit renal physiology accumulated over the years by our 

collaborator Dr. Charles Wingo, we selected the RCCT-28A cell line for use in our 

experiments. 

The RCCT-28A transformed cell line was derived from immunodissected rabbit CCD 
by Arend et al 1989. The RCCT-28A line was created by microdissecting cortical 
collecting tubule and dispersing cells on plates coated with monoclonal antibody specific 
to collecting tubule cells. The antibody coating the plates, known as IgG 3 (rct-30), had 
been made against rabbit renal cortical cells injected into mice. The resultant monoclonal 
antibody stained only collecting tubules on cryotome sections. Staining was primarily 
basolateral in intercalated and principal cells. Cells from the dissected rabbit collecting 
duct that had bound in the antibody-coated dish were immortalized with an adenovirus 
2-SV40 hybrid, and then cloned by limited diffusion. A population of cells that continued 
to proliferate while retaining epithelial morphology was obtained. 

The antigenic and hormone response of these cells is specifically consistent with their 
origin in the cortical collecting tubule. Immunocytochemistry showed reactivity in 100% 
of cells to an antibody (mr-mct) against mitochondria-rich cells of the medullary collecting 
duct (Schweibert et al, 1992). The mr-mct antibody was seen to be specific for 
acid-secreting, or type A cells (Burnatowska-Hledin and Spielman, 1988). 
Immunoreactivity was also observed to an antibody to band 3 protein (IVF12), another 
marker for acid-secreting intercalated cells (Schweibert et al, 1992). The presence of 
carbonic anhydrase in >95% of cells was indicated by the binding of a fluorescent 
acetazolamide analog (Dietl et al, 1992). As expected for an intercalated cell, 
conductance of CI", but not Na 1 or K', was indicated by patch clamp measurements (Dietl 



25 
etal, 1992). Schweibert et al. (1992) saw no antigenicity of the cells toward two 

antibodies specific for base-secreting (or type B) cells or toward four antibodies specific 

for principal cells. The origin, collection, and characterization of these cells indicates that 

they are a good model of the acid-secreting intercalated collecting duct cell. 

The first study undertaken using these cells showed that adenosine analogs increase 

intracellular calcium by stimulating phosphoinositide turnover (Arend et al., 1989). 

Inositol turnover was measured by labeling cells with myo-[ 3 H]inositol and detecting 

[TTJinositol phosphate formation. A 1 -receptor agonists increased phosphoinositide 

turnover, and the increase was blocked by an A 1 -receptor antagonist. Adenosine also 

regulated a 305 pS chloride channel in RCCT-28A cells via protein kinase C and a G 

protein (Schwiebert et al., 1992). Chloride channels in these cells have been characterized 

by the patch clamp method, showing CI' conductance to be stimulated by isoproterenol 

(Dietl etal., 1992; Dietl and Stanton, 1992). Cell swelling of RCCT-28A cells activated a 

CI" conductance by altering the organization of actin filaments (Mills et al., 1994; 

Schwiebert etal., 1994). Activation of the channel was mimicked by stretching the 

membrane and disruption of F-actin by dihydrocytochalasins. Stabilizing F-actin with 

phalloidin blocked activation of the CI' channel. Bello-Reuss (1993) reported H,K-ATPase 

activity in RCCT-28A cells, observing an apical acidification mechanism that had a 

component sensitive to withdrawal of K' and two H,K- ATPase inhibitors, Sch-28080 and 

omeprazole. She also detected activities suggestive of an apical H-ATPase and a 

basolateral C1/HC03 exchanger. Acid secretion by these cells was diminished in cells 

grown at low pCO : , evidence of regulation of the acidification mechanism by alkaline 

conditions. These cells have proven useful in studying various processes normally 



26 
associated with acid-secreting intercalated cells. The observation of H,K-ATPase activity 

in these cells made them particularly appealing for our studies. 

Summary 

H,K-ATPase activity in the collecting duct has very real implications for maintenance 
of health and well-being. Studies have shown a myriad of derangements in the absence of 
functional H,K-ATPase activity, including a real possibility of death. The H,K-ATPase 
provides an excellent example of how critical some of the enzymes are that fine tune the 
environment of the body. It is interesting that an enzyme that has such an immense level of 
activity in one organ, the stomach, actually is more important in another, the kidney, in 
which its level of activity might be called small. 

Several challenges exist in studying the H,K-ATPase enzyme and its function. One of 
these is the lack of knowledge of the quantitative effects of ouabain and Sch-28080 on the 
HKa 2 isoform of the enzyme. Another its low level of expression, making assays of 
mRNA, protein, and activity difficult. There was also a dearth of knowledge about the 
molecular forms of H,K-ATPase in the kidney when these studies were undertaken. This 
situation has changed, and the contributions described herein have been part of that 
change. 



MATERIALS AND METHODS 



Molecular Biology 



Tissue culture 

The RCCT-28A cell line was derived from immunodissected renal cortical collecting 
duct (Arend et cil., 1989). These cells were the kind gift of Dr. William Spielman, and 
experiments were performed using cells between passages 1 1 and 3 1 . Cells were grown in 
DMEM media supplemented to 10% with FBS and to 1% with Penicillin-Streptomycin. 
All media was bubbled overnight with a mixture of 5% C0 2 , 21% On, 74% N 2 , then filter 
sterilized by use of a 0.20 urn cellulose acetate syringe-mounted filter (Corning, Corning, 
NY). Cells were maintained in culture in tissue culture flasks at 37°C in a 5% C0 2 
atmosphere. Media was changed on alternate days, and cells were split 4: 1 at confluency. 
For experiments, cells were passaged to Corning Costar Transwell Collagen-coated 
semipermeable inserts. Cells were plated to a density of 2xl0Vcm 2 on the inserts, grown 
for 2 days in media containing 10% FBS, and then shifted to 0.1% FBS for a period of 24 
hr prior to the experiment. 



27 



28 
Isolation of total RNA 

Total RNA was isolated from cells or tissues employing the method described by 
Chomczynski and Sacchi (1987). After aspiration of media, tissue culture cells were rinsed 
in ice cold sterile PBS (10 mM sodium phosphate, pH 7.4, 150 mM NaCl). Next, 0.5 mL 
of GTC solution (4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% 
n-lauroylsarcosine, 100 mM P-mercaptoethanol) was added to promote cell lysis. The 
resulting viscous fluid was scraped from the insert and emptied into polypropylene 
centrifuge tubes on ice. New Zealand White Rabbits were sacrificed by decapitation and 
the kidneys removed immediately. Kidneys were dissected under a microscope, slicing 
coronally to separate cortex and medulla. Distal colon was prepared by clipping the distal 
25 mm of colon, cutting longitudinally, then rinsing away contents by a thorough spray of 
ice cold PBS. Gastric mucosa was collected by slicing the stomach transversely, rinsing in 
ice cold PBS, then scraping the rugae with a Scoopula (Fisher, Pittsburgh, PA). Tissues 
were dounce homogenized in 4 mL GTC solution until the suspension appeared to be 
homogneous. The resulting viscous fluid was poured into polypropylene centrifuge tubes 
on ice. 

While being held on ice, 1 volume phenol was added to the homogenized tissue, then 
1/10 volume 2 M sodium acetate (pH 4.0), and 0.22 volume chloroform-isoamyl mixture 
(24:1). After each addition, the samples were briefly vortexed. The samples were 
subjected to centrifugation at 1000G X 25 min at 4°C. The upper phase was retained, and 
RNA was precipitated twice in isopropanol and twice in ethanol, then resuspended in 100 
uL DEPC-treated water. Storage of the RNA was at -20°C. RNA concentration was 



29 
determined by OD :60 reading on a spectrophotometer. For absorbance of 1.0 at 260 nm, 

the concentration was taken to be 40 u,g/ml. 

Northern analysis 

Northern blots were done following the procedures of Sambrook et al. (1989). 
Samples of total RNA (20 ug per lane) or mRNA (2 ug per lane) underwent 
electrophoresis in a 1% agarose, 0.22 M formaldehyde denaturing gel. Capillary transfer 
to a nylon membrane (Hybond N, Amersham Corp., Arlington Heights, IL) was conducted 
overnight in 20X SSC (3M NaCl, 300 mM Na citrate). Absorbent paper towels were 
changed twice during transfer. RNA is immobilized on the membrane by baking 2 h at 
80 C in a vacuum oven. 32 P-labelled probes were prepared by random primer extension of 
75-150 ng DNA according to the protocol of the Megaprime Kit (Amersham). 
Membranes were prehybridized a minimum of 1 5 min and hybridized with labelled probe 
for 24 h in hybridization solution at 65°C. When heterologous probes were employed, such 
as cross-species probing, temperatures as low as 42°C were used to reduce stringency. 
Washing was done first at room temperature for 20 min in IX SSC, 0.1% SDS, then 3 
times at hybridization temperature for 20 min in 0.2X SSC, 0. 1% SDS. After washing, 
membranes were exposed to Kodak BioMax MS film at -80°C with Kodak BioMax 
intensifying screens. Exposures between three and six days were often required to detect 
H,K-ATPase subunit mRNA. An mRNA probe for glyceraldehyde-3 -phosphate 
dehydrogenase, a glycolytic enzyme, was used as a control to ensure even loading 
amounts of RNA between lanes. An exposure of eight hr was typically sufficient to 
visualize this control. 



30 
RT-PCR using degenerate primers 

RT- PCR was carried out as described by Davis el al, 1994. The cDNA template for 
the PCR reaction was produced by incubating 1 ug total RNA from microdissected rabbit 
renal cortex and 0. 1 ug random hexamers in a volume of 1 1 uL at 70°C X 2 min. The 
reaction mixture was quenched by placing the tube on ice. The reverse transcription 
reactions were carried out at 37°C X 1 hr in a volume of 25 uL. The mixture contained 
RNA-random hexamers mix in the final concentrations indicated: First Strand Buffer (50 
mM Tris-HCl (pH 8.3), 75 niM KC1, 3 mM MgCl : ), dithiothreitol (lOmM), dNTPs 
(2mM), RNAsin (30 U, Promega, Madison, Wl), and Superscript II (200 U, Gibco BRL, 
Gaithersburg, MD). PCR reactions were primed with pairs of oligonucleotides shown in 
Table 2-1 synthesized by the University of Florida Interdisciplinary Center for 
Biotechnology Research (UF ICBR) DNA Synthesis Core. PCR reactions were 
performed in a volume of 100 uL at the final concentrations indicated: dNTPs (0.22 mM), 
PCR Buffer (20 mM Tris-HCl, pH 8.4, 50 mM KC1), MgCI 2 (50 mM), primers (1 ug 
each), and Tag DNA Polymerase (5 U). The reactions were overlaid with 50 uL mineral 
oil. Thermal parameters of the reactions included a 5 min X 94°C presoak followed by 
94°C X 40 sec denaturation, 55°C X 1 min anneal, and 72°C X 2 min extension for a total 
of 30 cycles followed by a final extension of 5 min. Reactions were held at 4°C overnight 
before PCR product cloning into the pCR II vector and transformed into OneShot E. coli 
cells utilizing the TA Cloning Kit (Invitrogen, San Diego, CA). E. coli cells containing the 
plasmid of interest were grown overnight in a 5 mL culture and plasmids isolated using the 



31 
QiaPrep Spin Mini Kit (Qiagen, Santa Clarita, CA). Sequencing of plasmids was carried 
out by the UF ICBR DNA Sequencing Core. 

RT-PCR using standard primers 

A reverse transcription reaction was carried out as in the RT-PCR method described 
above for degenerate primers. PCR reactions were primed by pairs of oligonucleotides 
synthesized by the UF ICBR DNA DNA Synthesis Core and summarized in Table 2-1. 
Reactions were carried out using 1 ug total RNA in a 50 uL volume in KlenTaq PCR 
Reaction Buffer (40 mM Tricine-KOH, pH 9.2, 15 mM KOAc, 3.5 mM Mg(OAc) 2 , and 
75 ug/mL Bovine Serum Albumin), primers as listed above (0.5 uM), and dNTPs (0.8 
mM), and Advantage KlenTaq Polymerase Mix (Clontech, Palo Alto, CA). Thermal 
parameters of the reactions included a 2 min X 94°C presoak followed by 94°C X 30 sec 
denaturation and 68°C X 1 min extension with a final extension of 5 min. The numbers of 
cycles were specific to each experiment and are indicated in the 1 legends. PCR reactions 
were purified for sequencing by phenol/chloroform (1:1) extraction and two 3000 G X 5 
min centrifugation steps in Ultrafree-MC 30000 NMWL regenerated cellulose columns 
(Millipore, Bedford, MA). Due to low yield of the HKctl PCR reaction, a 45 cycle 
reaction was carried out to generate product for sequencing. Sequencing of PCR products 
was carried out by the UF ICBR DNA Sequencing Core. 

Genomic PCR 

To amplify genomic DNA for sequencing, template used was 1 ug Clontech Rabbit 
Genomic DNA. Clontech KlenTaq Advantage PCR Mix was used with components and 



32 



< 



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33 
concentrations specified in the description of RT-PCR above. Primers were 

ACCCGCGGCGCCTCCAGCGCGACAT (nucleotides 16-40, BC386) located in the 
first exon of HKa* and TATCTGTAGCTGCATGGTGCTCCAC (nucleotides 69-93, 
BC334) located in the second exon of HKotia. Thermal parameters of the reactions 
included a 1.5 min X 94°C presoak followed by 5 cycles of 94°C X 15 sec denaturation 
and 72°C X 2 min extension, 5 cycles of 94°C X 15 sec denaturation and 70°C X 2 min 
extension, and 25 cycles of 94°C X 15 sec denaturation and 68°C X 2 min extension with a 
final extension of 8 min. Reactions were held at 15°C overnight before ligation of the 
products into the pCR 2. 1 vector. The ligation mixture was transformed into OneShot E. 
coli cells utilizing the TOPO-TA Cloning Kit (lnvitrogen, San Diego, CA). E. coli cells 
containing the plasmid of interest were grown overnight in a 5 mL culture and plasmids 
isolated using the QiaPrep Spin Mini Kit. Sequencing of plasmids was carried out by the 
UF ICBR DNA Sequencing Core 

3' RACE 

A 3' RACE reaction was carried out as described by Davis el al, 1994. mRNA was 
prepared from rabbit renal cortex total RNA using the PolyATtract (Promega, Madison, 
WI) system. A reverse transcription reaction was carried out as in the RT-PCR method 
described above, using 1 ug mRNA as template and 0. 1 ug primer 
(GACTCGAGTCGACATCGA[T] 17 , BC229). The complementary strand was next 
synthesized using 5 uL of RT reaction along with 0. 1 ug of sense primer 
(TGCGGAAACTCTTCATCAGG, nucleotides 3088-3 107, BC262) in a reaction volume 
of 98 uL that contained the following components: PCR Buffer (20 mM Tris-HCl, pH 



34 
8.4, 50 mM KC1), MgCl : (50 mM), dNTPs (0.22 mM), overlaid with 50 uL mineral oil. 

After a 95°C incubation for 5 min, the temperature was lowered to 70°C and 5 U Taq 
DNA Polymerase was added. A 2 min annealing phase followed at 55°C, then the 
complementary strand was extended at 72°C for 10 min. Antisense primer (0. 1 ug, 
GACTCGAGTCGACATCG, BC230) was added, and a PCR reaction was initiated with a 
94°C X 40 sec denaturation, followed by a 55°C X 1 min anneal, and a 72°C X 2 min 
extension for a total of 40 cycles. Final extension was for a duration of 5 min. Reactions 
were held at 4°C overnight before cloning PCR products into the pCR II vector utilizing 
the TA Cloning Kit (Invitrogen, San Diego, CA) per manufacturer instructions. The 
library of clones produced in this manner was screened using an oligo 
(CTCTACCCTGGCAGCTGGTG, nucleotides 3 108-3 127, BC261) 5' labeled using the 
ECL kit (Amersham, Arlington Heights, 1L). Sequencing was carried out by the UF ICBR 
DNA sequencing core. 

5' RACE 

A 5' RACE reaction was performed using the Marathon cDNA Amplification Kit 
(Clontech, Palo Alto, CA) following manufacturer's instructions with the following 
modifications. RT reactions were carried out using 5.5 ug total RNA of rabbit renal 
cortex, incubated with the gene-specific primer TTGCCATCTCGCCCCTCCTT 
(nucleotides 121-102, BC33 1) for 30 min at 50°C, then at 55°C for 1 5 min. PCR reactions 
were carried out using the anchor primer (CCATCCTAATACGACTCACTATAGGGC, 
API) included in the Marathon kit paired with gene-specific primer 
TATCTGTAGCTGCATGGTGCTCCAC (nucleotides 93-69, BC334). Concentrations 



35 
and components of the reactions were detailed above for use of Clontech KlenTaq 

Advantage Polymerase Mix. Thermal parameters of the reactions included a 1 min X 94°C 

presoak followed by 5 cycles of 94°C X 15 sec denaturation and 72°C X 1 min extension, 

5 cycles of 94°C X 15 sec denaturation and 70°C X 1 min extension, and 25 cycles of 94°C 

X 15 sec denaturation and 68°C X 1 min extension with a final extension of 8 min. 

Evaluation of PCR products 

PCR products were analyzed by agarose gel electrophoresis. 20 u.L of reactions were 
run along with 1 mL DNA loading dye (50% glycerol, 1% xylene cyanol, 1% 
bromophenol blue) on a 1.2% agarose gel in TAE buffer (Tris mM, acetic acid mM, 
EDTA mM). Low DNA Mass Ladder (Gibco BRL) was electrophoresed alongside PCR 
products to quantitate DNA concentration, and 100 bp ladder (Gibco BRL) to evaluate 
the size of the products. 

In some cases, products were not visible after ethidium bromide staining, so Southern 
blotting was used to visualize the products. Procedures followed were as described by 
Davis et ai, (1994). After running products on gels as described above, gels were soaked 
30 min in denaturation solution (1.5 M NaCl, .5 M NaOH), and 30 min in neutralization 
buffer (1 M ammonium acetate, .02 M NaOH). Capillary transfer in neutralization solution 
and baking 80° X 1 hr was utilized to adhere DNA to nylon membrane (Hybond N). 
Probes to hybridize specifically to the expected products (Table 2-1) were created using 
RT-PCR with rabbit renal cortex as template, cloning the inserts using the TA Cloning Kit 
as described above. Inserts were sequenced to confirm their identity. Probes were labelled 
using the ECL Direct System(Amersham) following manufacturers instructions. Exposure 



36 
to Hyperfilm ECL (Amersham) for a period of two hr was required to visualize the results 
of PCR reactions. 

Biochemistry 

Preparation of membrane protein from tissue culture cells 

Two clusters of six wells each were used for each experimental condition; this 
typically yielded 50 pg of protein. All solutions and glass douncers are cooled on ice, and 
centrifuges are either refrigerated or located in cold rooms. Cells are held on ice at all 
times. Cells were rinsed in PBS containing 0.5 mM PMSF, 1 .5 mL in top well of 
Transwell, 2.5 mL in bottom well. Following aspiration of the rinse solution, to each 
single well was added 0.5 mL PBS, 0.5 mM PMSF, 1 mM EDTA. A cell scraper was used 
to dislodge cells from the insert. This solution containing cells was pipetted from one well 
to the next dislodging and gathering all the cells from one cluster. This was repeated with 
a fresh 0.5 mL solution on the same cluster to remove any remaining cells. The two passes 
combined for a total of 1 mL, and the other clusters were processed in the same manner. 

Cells were spun in a centrifuge for 5 min at 500 X g to remove cellular debris. 
Supernatant was discarded, and the pellet was resuspended in swelling buffer (tris 10 mM 
pH 7.8, 1 mM EDTA, 1 mM PMSF, 2 pM aprotinin, 2 uM leupeptin, 2 pM pepstatin) for 
15 min. Cells were homogenized by 50 strokes in a glass douncer. After moving cells back 
to a microfuge tube, 0. 1 1 mL of 10X salts (300 mM NaCl, 20 mM MgCI 2 , 10 mM Tris 
pH 7.8) was added before vortexing. Mixture was spun in a centrifuge for 1 minute at 
1000 X g to remove nuclei. Supernatant was retained and spun in a centrifuge for 5 min at 



37 
1500 X g. Supernatant was retained and spun in a centrifuge for 30 min at 23000 X g. 

Cells were resuspended in 20 u.L resuspension buffer (1 volume swelling buffer, 1/10 

volume 10X salts) and stored at -20°C. 

Preparation of membrane protein from rabbit tissues 

Kidney, distal colon, and stomach tissues were obtained in the same manner as for 
RNA isolation. Care was taken to maintain solutions and apparatus ice cold. Two rabbit 
distal colons were required per preparation to yield useful concentrations of protein. 
Tissues were homogenized for 15 seconds at 12500 rpm (setting 8 on Omni-Sorvall tissue 
homogenizer) in Buffer A (50 mM sucrose, 10 niM Tris pH 7.4, 1 mM EDTA, . 1 mM 
PMSF). After allowing 15 seconds for settling, homogenization was repeated. Three 
volumes buffer B (250 mM sucrose, 10 mM Tris pH 7.4, 1 mM EDTA, . 1 mM PMSF) 
were added, then homgenate spun in a centrifuge for 10 min at 1000 X g. The supernatant 
was subjected to centrifugation for 20 min at 10000 X g three times. Final centrifugation 
was for 1 hr at 100000 X g. After discarding supernatant, pellet was resuspended in 500 
UL loading solution (1 mM Tris, 10 mM MgCb, 150 mM NaCI). After transferring 
resuspended pellet to glass douncer, dounce is dropped into the douncer, turned three 
times, then raised and dropped again. This was done ten times. The resultant preparation 
was stored at -20°C. 

Antibodies 

Peptides used as immunogens were designed for maximum antigenicity and minimum 
homology to other proteins. Avoiding homology to other P-type ATPases was particularly 



38 
important. Rabbit H,K-ATPase catalytic subunit sequences were scanned using the 

computer program PEPTIDESTRUCTURE (Genetics Computer Group, 1997) to find 
regions relatively high in charged, hydrophilic residues (Jameson and Wolf, 1988). Such 
regions were then searched by BLAST (Altschul et a/., 1990) to eliminate those that could 
be predicted to cross-react with other proteins. Once these constraints were met the 
candidate sequences were examined using the program MOTIFS (Genetics Computer 
Group, 1997; Bairoch and Apweiler, 1996) to ensure that there were no potential sites for 
protein modification that might affect reactivity. In the case of HKai and HKct 2a , there 
was only one region that satisfactorily met all of these criteria. In the case of HKa 2c , there 
were two, one being the region in common with HKa 2a , the other in the extended amino 
terminal region of HKct 2c not contained in HKa 2a . 

Peptides were synthesized with an N-terminal cysteine and conjugated to keyhole 
limpet hemocyanin (UF ICBR Protein Core) using the Pierce (Rockford, IL) Imject 
system. Three peptides were used as immunogens, the first was designed to react with a 
portion of HKa,, the second was designed to recognize a portion of HKot 2 found in both 
HKa 2a and HKa 2c , the third to a portion unique to HKa 2c . The peptide chosen for HKa, 
corresponded to amino acids 569-582 (CLYLSEKDYPPGYAF). The peptide chosen 
within the common region contained amino acids 18-37 in HKa 2a and 79-88 in HKa 2c 
(CDIKKKEGRDGKKDNDLELKR). The peptide chosen within the HKa 2c -specific 
region corresponded to amino acids 13-25 (CGEERKEGGGRWRA). Antipeptide 
antibodies were raised in chickens by Lofstrand Laboratories (Bethesda, MD). Chickens 
received boost innoculations at 21 day intervals, and were exsanguinated at day 73 to 



39 
produce antisera. Preimmune sera was collected prior to initial innoculation. Yolks of eggs 

collected over the two week period prior to final bleed were pooled, and immunoglubulins 

purified from yolk material by the Promega EGGstract method. Concentration of the 

EGGstracted yolks was determined by the modified Lowry procedure of Markwell et al. 

(1978) and the concentrations adjusted to 2 mg/mL by addition of IgY buffer solution 

(Promega). Purity and concentration of the IgY obtained was confirmed by non-reducing 

SDS-PAGE and staining with Coomassie blue. 

Western analysis 

Protein concentrations in tissue and cell samples were determined by modified Lowry 
(Markwell et al, 1978). Proteins (10 pg/lane) were separated on 4-20% reducing 
SDS-polyacrylamide gels (BioRad, Hercules, CA ), 10 pg per lane. Vesicle preparations 
were suspended in buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 5% 
p-mercaptoethanol, 3% SDS) and incubated 2 min X 90°C prior to electrophoresis. Gels 
were rinsed 10 min in TBS (10 mM Tris- HCI pH 7.2, 150 mM NaCI), and 10 min in 
transfer buffer (20 mM Tris-HCL, 150 mM glycine, 20% methanol, pH 8.3). Proteins 
were electrotransferred at 104 V, .25 A, 4°C to Hybond ECL nitrocellulose membranes 
(Amersham) in transfer buffer. Blocking of membranes was done in TBS-T (TBS, 0. 1% 
TWEEN-20) containing sodium azide and 5% non-fat dry milk at 4°C overnight or room 
temperature for 1 hr. Antibody incubations were one hour each, carried out in TBS-T 
containing 5% non-fat dry milk. Following blocking and following primary and secondary 
antibody incubations, immunoblots were rinsed in TBS-T with continuous agitation. This 
was done three times for one minute each, then twice for 5 min each. Primary antibodies 



40 
purified from egg yolk were used at a dilution of 1 :200, the anti-chicken IgY-horseradish 

peroxidase conjugated secondary antibody (Promega) was diluted to 1 : 10000. When 

antisera or preimmune sera were used as primary antibodies, dilution was 1 :2000. A final 

wash was carried out in TBS for 10 min, and then antibody reactivity was detected using 

chemiluminescence (Pierce). Apparent molecular masses were established using the High 

Mass Range Molecular Weight Markers (BioRad). 

Fluorescence Microscopy 
Measurement of pH, 

The fluorescent, pH-sensitive dye BCECF-AM was used to directly measure pH, in 
RCCT-28A tissue culture cells. BCECF-AM was stored as a stock at -20^ in a 30 mM 
solution in DMSO. Cells were incubated at room temperature for a period of 30 min in 
solution 1 (Table 2-2) with BCECF-AM at a final concentration of 5 //M. A minimum of 
5 min perfusion with solution I delivered at 37°C was allowed to rinse BCECF away at 
the beginning of each experiment. 

Cells were imaged by epifluorescence at 530 nm emission on an inverted microscope 
(Boyarsky et al, 1988, Weiner and Hamm, 1989) using excitation wavelengths of 440 nm 
and 490 nm. These wavelengths correspond to the isosbestic point and to a highly 
pH-sensitive wavelength of BCECF, respectively. The ratio of emission intensities at the 
two wavelengths is directly proportional to pH over the pH range being studied, and is 
constant with respect to such variables as cell-to-cell variations in dye uptake and leakage. 
A field containing approximately 50 cells could be visualized using a Nikon X40, 0.55 



Table 2-2. Solutions for determination of pH, 



Solutions 



41 



NaCl 

Choline CI 

Ammonia CI 

KC1 

KH 2 P04 

Phosphoric acid 

Na-acetate 

Acetic acid 

CaCl 2 

MgS0 4 

Alanine 

Glucose 

HEPES 



19.2 



102.2 122.2 99.2 



102.2 122.2 119.2 



20 



> .-> 



25 25 



2U 



25 



25 



25 



20 
3 



12 1.2 1.2 1.2 1.2 1.2 1.2 



8.3 8.3 






25 



25 



Note: Concentration units are in mM. Osmolality was adjusted to 290±5 mosmol/kg H 2 
by addition of the principal salt. pH was adjusted to 7.40±05 by addition of 
tetramethylammonium-OH. Solutions were bubbled with 100% : . 



42 
LWD lens on a Nikon Diaphot-TMD inverted microscope. Excitation light was provided 
by a 100 W mercury lamp. The light was split into two beams by a 470 nm low-pass 
dichroic mirror. The two split light beams then passed through filters to yield beams of the 
desired wavelengths. The transmitted light path contained a 440 nm filter; the reflected 
light path contained a 490 nm filter. Computer-actuated shutters on each light path 
alternated the incident light wavelengths and minimized the time the cells were subjected 
to the high intensity light. The two light beams were recombined by a second 470 nm 
low-pass dichroic mirror. Light was directed to the microscope stage by a 510 nm 
high-pass mirror, and the emitted light was directed to a Videoscope KS-1381 image 
intensifier coupled to a Dage 72 CCD camera. Because the optics necessary to image cells 
grown on inserts required relatively intense incident light, measurement frequency was 
limited to avoid phototoxicity. Therefore, measurements were made at 30 second 
intervals. Images were digitized and stored by computer allowing subsequent analysis of 
single cells using the Image 1/FL software package (Universal Imaging Corp., 
Westchester, PA). 

During measurements, cells were constantly perfused at a rate of -10 mL/min by 
HEPES-buffered solutions that were continuously bubbled by 100% 2 and heated to be 
delivered at a temperature of 37°C. Switches for the various solutions were located 
physically near the input to the cell chamber to minimize the time required to switch 
solutions, and fluid was continuously removed from the opposite side of the chamber by 
vacuum suction. For some experiments, the apical side of the cells was perfused by 
different solutions than the basolateral side of the cells by utilizing separate input tubes in 
the upper and lower chambers of the Transwell inserts. 



43 
Cells were acid-loaded using the NH 4 C1 prepulse technique. Briefly, cells were 

incubated with 20 mM NH4CI (equimolar substitution for NaCl) for 5 min, then 
ammonium chloride was removed from the perfusing solution. Addition of inhibitors or 
K + removal began at the start of the ammonium pre-pulse. The ethylisoproplamiloride 
(EIPA) stock solution was 1 mM in dimethyl sulfoxide (DMSO), and Sch-28080 was 10 
mM in DMSO. EIPA was obtained from Research Biochemicals, International (Natick, 
MA) and Sch-28080 was the kind gift of Dr. James Kaminski at Schering Corporation 
(Bloomfield, NJ). Inhibitors were stored as stock solutions at -20°C and diluted into the 
solutions indicated immediately preceding their use. 

Calibration of the pH, measured by BCECF fluorescence was carried out by the 
nigericin calibration technique of Thomas el al. (1979). Calibration solutions (120 mM 
KC1, 1.2 mM CaCl 2 , 1 mM MgCl : , 25 mM HEPES, 14 uM nigericin) were adjusted to 
6.6, 6.8, 7.0, 7.2, and 7.4 pH units using 1 M NaOH and HC1. Cells were incubated with 
each solution for a minimum of three min, and three fluorescence ratio measurements were 
taken at each pH. 

Statistical methods 

pH, recovery rates for individual cells were calculated using least-squares linear 
regression. Rates were calculated for the period beginning two min after NH4CI 
withdrawal, allowing time for cells to equilibrate after acidification, and ending with Na + 
addition or EIPA withdrawal. Data were collected for independent experiments involving 
separate passages of cells. pH, recovery rates for each experimental condition were 
presented as the mean of the rates determined for the individual experiments ± SE. Cells 



44 
without NaVH* exchanger activity, defined as an increase in pH, recovery with EIPA 

removal, were classified as non-viable and excluded from further analysis. P<0.05 by 

Student's Mest was taken as significant. 



H,K-ATPASE 3 SUBUN1TS IN THE RABBIT RENAL MEDULLARY COLLECTING 

DUCT 

Introduction 



There are multiple H,K-ATPase subunit isoforms that are involved in coupled H + for 
K + active exchange in the kidney. These now include the NaK(3i subunit that is the newly 
recognized partner for the HKa 2a subunit, in addition to its long-known role as partner to 
NaKcti. In addition to the complexity involved in having multiple H,K-ATPase isoforms 
present, there is the possibility of variant transcripts of each H,K-ATPase subunit. 
Differences in polyadenylation sites, transcription start sites, and alternative splicing in a 
tissue-specific manner might further complicate the picture. 

When rat HK(3 subunit was originally sequenced, multiple transcriptional start sites 
were observed by primer extension experiments (Newman and Shull, 1991). It was not 
known what role these various transcriptional start sites play. When the Cain, Wingo and 
Nick laboratories did preliminary studies concerning the regulation by K + status of the 
HK(3 subunit in rabbits, it was noted that on some northern blots of renal tissues HKP 
subunit mRNA appeared as a doublet. This was evidence for a second H,K-ATPase 
subunit transcript present at a level comparable to the primary transcript. 

Here we show that there is a variant HKp subunit mRNA that is expressed in renal 
medulla, and not in renal cortex or stomach. We have designated the variant mRNA 

45 



46 
HKP'. The HKP' transcript is found in medulla at a level comparable to the quantity of 

the HKP subunit, and the translation start site is unchanged, so both HKP and HKP' 

encode the same protein. HKP' may be the product of alternative splicing. 

Renal Medulla HKP mRNA Variant 

In order to investigate the doublet band in northerns probed with Hkp cDNA probes, 
RNA was prepared from stomach, renal cortex, and stomach. In northern analyses (Figure 
3-1) involving twenty different rabbits in experiments spaced over several years, only 
single transcripts were observed in either gastric or renal cortical tissues. Gastric and renal 
cortical transcripts appeared to be of identical size. However, in renal medullary tissues, a 
doublet was universally visible. Northern analysis showed that the smaller of the two 
medullary HKP transcripts was the same size as in the other two tissues, and the second 
renal medullary transcript was larger. The quantity of the second mRNA, HKP', was 
generally comparable to the quantity of H1<P mRNA. 

In order to determine the molecular nature of the different transcripts of the medulla, 3' 
and 5' RACE experiments were conducted using mRNA isolated from rabbit renal 
medulla. The rabbit RNA was selected from those that showed prominent HKP subunit 
upper bands by northern analysis because these would have a higher abundance of the 
novel transcript. Representative PCR products generated by the 3' and 5' RACE 
experiment are shown in Figure 3-2. PCR often 3' RACE products sequenced, all were 
the same, and all extended to within 5 bp of the published HKP cDNA sequence (Reuben 
etal., 1990). Ten 5' RACE products were also sequenced. Nine of these were similar to 















47 






HK(3 



m 

■ :. ■ ..v. 




49 - 1-4 kbp 









GAP3DH 







- 1.3 kbp 






■ 



3 

o 

o 

a 

x 






I 

3 

| 

ST 



C/3 



o 



Figure 3-1 . Northern analysis showing presence of HK0 mRNA in renal cortex, renal 
medulla, and stomach. The existence of two mRNA species was clearly visible in renal 
medulla, whereas a single mRNA species was detected in stomach and renal cortex. 
GAP3DH was used to show the condition of the RNA samples. 









48 



750 bp - 





• 


U) 


KM 


i 


i 


o 


o 


w 


w 



Figure 3-2. 3' and 5' RACE reactions to amplify HKp cDNAs using rabbit renal 
medulla RNA as template. 



49 
the published HKp cDNAsequence. However, all ended approximately 20 bp from the 5' 

end of the published sequence making it impossible to confirm the extreme 5' end of the 
sequence. 

One of the sequences had a very different 5' end than the others. In that sequence 
(Figure 3-3 A), the first 1 1 bp of the published HKP sequence was not found, and in its 
place there was a different 1 1 8 bp region (Figure 3-3B). The sequence was confirmed by 
5' RACE using a primer specific to the variant extension 
(CCCTGCACCCCGACTGAGG, nucleotides 104-121, BC388). 

To examine the possibility that this renal medulla-specific transcript might be regulated 
by K + status in rabbits, northern analysis was carried out using total RNA from animals fed 
a low K + diet. Renal medulla total RNAs from four rabbits fed a Harlan (Indianapolis, IN) 
control diet and four rabbits fed a Harlan K -restricted diet for two weeks were probed to 
visualize HKP subunits using a probe made from 570 bp of coding region (Table 2-1). 
Neither of the two HKP transcripts of renal medulla had systematic variation in HKP 
mRNA level due to K + restriction in these animals. Northern analysis of renal cortex and 
stomach RNA from these same rabbits showed uniform level of HKp mRNA between 
rabbits. No regulation of HKp was observed, although in renal medulla there was a great 
deal of individual variation between rabbits. 

Discussion 

A rabbit renal medulla-specific HKp variant mRNA was cloned by 5' RACE and 
sequenced. It was not known why only one out of the ten sequences proved to be a 



A) 



50 



1 GGAGCTGATGGCTGCTGCTGATAGCACCGCCTCGAGCCAGCCCTGCAGGCGTCGCCCGCGATGCCTTTGACCGTGGCCGG 8 
+ + + + + + + 

81 GGGAGGCTATAAGACCCAGGGGGCCTCCGGCCTCAGTCGGGGTGCAGGGTGGGGGAGCGGCGGCTTCCACAGCAGACACC 160 
+ + + + + + + + 

161 ATGGCCGCCTTGCAGGAGAAGAAGTCGTGCAGCCAGCGCATGGAGGAGTTCCGCCACTACTGCTGGAACCCGGACACGGG 24 
+ + + + + + + + 

1MAALQEKKSCSQRMEEFRHYCWNPDTG 27 

241 ... 
2 8 ... Remainder of sequence omitted for clarity 



B) 



HKp 



HKp 



118 bp 




..ATG.. 



mRNAs 



Figure 3-3. HKp and p' subunits. A) The 5' end of the HKP' cDNA and its deduced 
amino acid sequence. Numbering begins at the start of the HKp' transcript. HKP' 
nucleotide 119 corresponds to nucleotide 12 of published gastric HKp sequence 
(Reuben, et al, 1990), and the point of divergence is marked byf . B) Comparison of 
HKp and HKP' mRNAs. There is no difference in the deduced amino acid sequence. 



51 
variant, since both transcripts are observed by northern analysis at comparable levels. 

Perhaps the extra length or RNA secondary structure reduced amplification efficiency. 
Because the variant 5' end differed in sequence from the 5' end reported for rabbit 
(Reuben et ah, 1990), the variant is presumed to arise from alternative splicing. This was 
not pursued because our focus was on the H,K-ATPase a isoforms. HKP mRNA and 
HKP' mRNA produced identical HK(3 subunit proteins. The splice sites of the related rat 
and mouse HKP genes (Newman and Shull, 1991 ; Canfield and Levenson, 1991) are not 
conserved, and working out this issue might have been a considerable target for our 
primary emphasis. 

The deduced amino acid sequence is unchanged by the variant 5' end. Differential 
regulation by alternate promoters may be the functional reason for the two transcripts. K + 
restriction was studied as a possible stimulus for regulating the promoters, but K + status 
alone did not correlate with differential regulation of the two transcripts. Other stimuli, 
such as aldosterone levels, may regulate these transcripts in renal medulla. It is interesting 
to note that although HKP subunit is expressed at extremely high levels in gastric tissues, 
this variant transcript was not observed in stomach. The tissue-specificity of the variant is 
striking. Study of its promoter region may be fruitful in terms of finding the cis acting 
factors that turn the gene on specifically in renal medulla. 



H,K-ATPASE a SUBUNITS IN THE RABBIT RENAL CORTICAL COLLECTING 

DUCT 

Introduction 



H,K-ATPase has been shown to play a role in acid/base and K + transport in kidney 
collecting duct (for review, see Wingo and Cain, 1993; Wingo and Smolka, 1995). 
However, many questions remained to be addressed, such as finding which H,K-ATPase 
isoforms are present in kidney, and determining their distribution along the nephron and 
collecting duct. This included defining the cell type specificity of each isoform. For many 
years, the rabbit has been the archetypal experimental animal for use in experiments 
involving microperfusion of renal tubules. To take advantage of the extensive knowledge 
of rabbit renal physiology in exploring H,K-ATPase function at the molecular level, 
primary structure information was needed for H,K-ATPase subunits in that species. 
Because of the discovery of H,K-ATPase catalytic subunit isoforms in addition to the 
gastric isoform in several species, the relationship between these isoforms needed to be 
examined. Perhaps most importantly, the role that the multiplicity of H,K-ATPase subunit 
isoforms plays in kidney function should be examined. 

At the time the experiments described here were undertaken, little was known of the 
identity of H,K-ATPase subunits in the kidney. mRNAs encoding catalytic subunit 
isoforms cloned from human axillary skin (Modyanov et al, 1991) and rat distal colon 

(Crowson and Shull, 1992) were the first to be detected in kidney. In collaborations 

52 



53 
including the Cain and Wingo laboratories, HK.P (Callaghan el a/., 1995, 

Campbell-Thompson et a/., 1995) was observed in rabbit kidney. The key experiment 
identifying HK0 in the kidney dated from 1992, prior to the arrival of this author, so that 
information was available in designing this dissertation project. As this work progressed, it 
was found that the HKoti subunit isoforms are present in renal tissues (Ahn and Kone, 
1995). In addition, an alternatively spliced isoform of rat HKct: was recently reported in 
kidney (Kone and Higham, 1998). The rat HKct: alternatively spliced variant was 
designated HKobb, with the original renamed HKa-,. The previous experiments were not 
designed to discriminate between HKa-, and HKa 2b . 

In these studies a full-length cDNA sequence was determined for the HKa : subunit in 
rabbit. An alternatively spliced variant of this HKa : subunit isoform was found in rabbit. 
The pattern of splicing was identical to that found in rat (Kone and Higham, 1998). 
However, the translation start site was not conserved. For this reason, the alternatively 
spliced variant in rabbit was designated HKa 2t . Antipeptide polyclonal antibodies were 
raised and used to show expression of both HKa : , and HKa 2c proteins in rabbit renal 
cortex. These results allude to a potentially complex pattern of regulation of H,K-ATPase 
activity in renal cortex. 

Multiple H.K-ATPase a Subunits in the Kidney 

We set out to find the HKa subunits that mediate the H,K-ATPase activities that had 
been observed in kidney. Because HKa-, mRNA had been observed in kidney, it was 
necessary to find the rabbit sequence of HKa 2: , in rabbit for use in designing tools for 



54 
studying H,K-ATPase function in the kidney. Rabbit HKcci sequence was known 

(Bamberg et al., 1992). In designing an experimental approach to this goal, we wanted to 
consider 1) the possibility that HKct] was expressed in kidney (unknown at the time) in 
addition to HKa 2 , 2) the possibility that the HKa 2 mRNA might be different in kidney than 
in the other tissues for which full length sequence was known, and 3) that novel HKa 
isoforms might be present in renal tissues. A good technique to address all these 
possibilities was to do RT-PCR using degenerate primers designed to anneal to regions of 
sequence that were highly conserved among P-type ATPases. This approach was expected 
to amplify any P-type H,K-ATPase that might be present in rabbit kidney. 

The aspartyl residue that is phosphorylated as an intermediate in the catalytic cycle of 
H,K-ATPase lies within a motif that is highly conserved in the entire P-type ATPase 
family. Another well-conserved region among P-type ATPases surrounds a lysine residue 
that can be modified by FITC. F1TC competes with ATP for binding (Jackson et al., 1983; 
Farley and Faller, 1985), so this region is thought to make up part of the ATP binding site. 
Advantage was taken of these two regions to design degenerate primers to amplify any 
P-type ATPase using renal cortex RNA as template. Fifteen P-type ATPase sequences 
were aligned using the computer program P1LEUP (Genetics Computer Group, 1997). 
These sequences were selected to give a wide range of mammalian Na,K- ATPases and 
H,K-ATPases. Chicken Na,K-ATPases were included because all three a subunit isoforms 
were known for that species so they comprised a good example of P-type ATPases from 
an organism less related to mammals. Toad sequences were included because toad 



55 
H,K-ATPase was one of only three non-gastric H,K-ATPases known at the time. 

Degenerate primers were then designed to amplify any of these ATPases (Figure 4-1). 

The products of the first two RT-PCR reactions using these primers, when cloned and 
sequenced, had high homology to the NaKoti subunit in other species. The third reaction 
product (Figure 4-2) was more related to the non-gastric H,K-ATPase a subunits than to 
the FEKcti subunit or to any of the Na, K-ATPase a subunits. It was a 419 bp fragment 
that corresponded to nucleotides 1 182-1600 of the full-length rabbit HKa 2l , cDNA 
sequence. A BLAST search listed three non-gastric H,K-ATPases as the most highly 
aligned sequences to the 419 bp sequence, with other H,K-ATPases and Na,K-ATPases 
being less well aligned (Figure 4-3). The sequence of the fragment shared 
89% nucleic acid identity with the rat distal colonic H,K-ATPase a subunit, 88% with a 
guinea pig distal colonic H,K-ATPase a subunit, and 86% with the H,K-ATPase a subunit 
cloned from human axillary skin. Based on these similarities, this fragment was tentatively 
identified as belonging to an H,K-ATPase. 

Using degenerate primers designed to anneal to conserved sequences we identified 
farther 5' and 3' in the sequence alignment paired with gene-specific primers designed 
based on the new sequence (Table 2-1), fragments containing more of the transcript were 
cloned (Figure 4-4). Outside the coding sequence, homology between cDNA sequences 
declines. With much of the coding region cloned and sequenced, 5' and 3' RACE were 
used to clone the full extent of the cDNA including the two ends. The 3' end was easily 
obtained; a single 3' RACE product was observed the first time the procedure was 
performed. The 5' end proved far more interesting, but more challenging to find. Initial 



A) 



56 



Hsu02076 

Ratatpasez 

Bmhkatpas 

Doghkatp 

Pigatphk 

Ocatprna 

Ratatpast 

Chknakat2 

Chknakat3 

Ratatpa3 

Ratatpa2 

Ratatpal 

Chkatpas 

Bmnkaal 

Tcatpmr 



CCATCATCTGCTCGGACAAGACTGGGACAC . 
CCATCATCTGCTCAGACAAGACGGGGACCC . 
CCATTATCTGCTCCGACAAAACAGGAACCC . 
CAGTGATCTGCTCAGACAAGACAGGGACCT . 
CAGTCATCTGCTCTGACAAGACGGGGACCC . 
CGGTGATCTGCTCCGACAAGACGGGGACCC . 
CAGTCATCTGCTCAGACAAGACAGGAACTC . 
CCACCATCTGCTCCGACAAAACCGGGACCC . 
CCACCATCTGCTCCGATAAGACCGGGACCC , 
CCACCATCTGCTCCGACAAGACCGGCACCC . 
CCACCATCTGCTCGGACAAGACAGGCACCC . 
CCACCATCTGCTCCGACAAGACTGGAACTC . 
CCACCATCTGTTCTGACAAAACAGGCACCC . 
CCACCATCTGCTCTGACAAGACCGGAACCC . 
CAACCATTTGCTCAGACAAAACTGGAACCT . 



Primer 



B) 

HSU02076 

Ratatpasez 

Bmhkatpas 

Doghkatp 

Pigatphk 

Ocatprna 

Ratatpast 

Chknakat2 

Chknakat3 

Ratatpa3 

Ratatpa2 

Ratatpal 

Chkatpas 

Bmnkaal 

Tcatpmr 



A A 
ATCTGCTCCGACAAAACCGG 
G G G 
T T 



. TCATGGTGATGAAGGGGGCCCCTGAGCGCA . 
. TCGTGGTGATGAAAGGAGCCCCTGAGAGGA . 
. TGCTCGTCATGAAAGGTGCCCCAGAGAGAA . 
. TGCTGGTGATGAAGGGCGCCCCCGAGCGCG . 
. TGCTTGTGATGAAGGGCGCCCCCGAGCGCG . 
. TGCTGGTGATGAAGGGCGCCCCCGAGCGCG . 
. TGCTGGTGATGAAGGGCGCCCCAGAGCGCG . 
. TCCTGGTGATGAAAGGGGCCCCCGAGCGCA . 
. TGCTGGTGATGAAAGGCGCCCCGGAGCGCA . 
. TGTTAGTGATGAAGGGCGCCCCTGAACGCA . 
. TGCTGGTGATGAAAGGTGCCCCGGAGCGCA . 
, TGCTAGTGATGAAGGGCGCCCCAGAAAGGA . 
, TGCTGGTGATGAAGGGAGCTCCAGAGAGGA . 
, TGCTGGTCATGAAGGGCGCCCCCGAGAGGA . 
, TGTTGGTGATGAAGGGAGCACCAGAACGGA . 



3 ' Primer 



A A 
GTGATGAAAGGCGCCCCCGA 
G G G 
T T 



Figure 4-1 . Design of degenerate primers for RT-PCR of novel P-type ATPases. A) 
Upstream primer was designed to anneal to well-conserved sequence at the enzyme active 
site phosphorylated aspartyl residue. GenBank loci of 1 5 aligned sequences are shown at 
left. Oligonucleotide is shown beneath aligned sequences. B) Downstream primer was 
designed to anneal to well-conserved sequence at the putative ATP binding site. 
Oligonucleotide is reverse complement of consensus sequence shown. 



57 



419 bp 




% 


3 

1 


OQ 




hd 


GO 




n 


O 




& 






t3 


C6 




!-t 






o 






Q* 






C 






o 






r-h 




■ 







Figure 4-2. RT-PCR product amplified from rebbit renal cortex RNA using degenerate 
primers. Gel isolated product was cloned and sequenced. 






58 



Sequences producing High-scoring Segment Pairs: 



gb U02076 HSU02076 
gb M90398 RATATPASEZ 
gb U94912 RNU94912 
db] |D21854|GPIHKAAS 
gb U94913|RNU94913 
emb| Z2 5809 | BMHKATPAS 
gb | M59960 | CHKNAKAT3 
emb | X05883 | RNATPAHO 
gb M14513 RATATPA3 
gb M28648 RATNALPH2 
emb | Z11798 | BMNKAA1 
gb 
gb 
gb 
gb 



U10108 
U49238 
J02649 
U17249 



XLU10108 
XLU49238 
RATATPAST 
XLU17249 



emb | X64694 | OCATPRNA 



gb 
gb 
gb 
gb 
gb 



J03230 
S66043 
U17282 
L42565 
L11568 



CHKATPAS 

S66043 

MMU17282 

HUMATP1G04 

DOGHKATP 



emb X02813 OAATPMR 



gb 

gb 

gb 

gb 

gb 

emb 

emb 

emb 

gb 

gb 



M22724 
M28647 
S74801 
M14511 
M74494 
X76108 
X05882 
X02810 
M59959 
U16798 



PIGATPHK 

RATNALPH1 

S74801 

RATATPA1 

RATNAKATP 

AASPAA 

RNATPAR 

TCATPMR 

CHKNAKAT2 

HSU16798 



emb | X04297 | HSATPAR 



gb 
gb 
gb 



J03007 
L42173 
M38445 



HUMATPAS 

DOGNKAA 

PIGATPBSEN 



emb | X03938 | SSATPAR 
gb|M14512|RATATPA2 
emb X58629 CCNAKATP 
emb X56650 AFNAKATP 
gb M75140 HYDATPASE 



gb 
gb 
gb 
gb 



L42566 
S76581 
J05451 
M63962 



HUMATP1G05 
S76581 
HUMATPGG 
HUMHKATPC 



Human ATP-driven ion pump (ATP1AL1.. 
Rat H+,K+-ATPase mRNA, complete cds. 
Rattus norvegicus H-K-ATPase alpha. . 
Guinea pig mRNA for distal colon H. . 
Rattus norvegicus H-K-ATPase alpha. . 
B.marinus mRNA for H,K-ATPase 
Chicken Na,K-ATPase alpha-3-subuni. . 
Rat mRNA homologous to alpha subun. . 
Rat Na+, K+-ATPase alpha(III) isof.. 
Rattus norvegicus Na,K-ATPase alph. . 
B.marinus mRNA for Na, K-ATPase al.. 
Xenopus laevis Na+-K+-ATPase alpha.. 
Xenopus laevis adenosine triphosph. . 
Rat stomach (H+, K+ ) -ATPase mRNA, c. 
Xenopus laevis gastric H ( +) -K(+ ) -A. . 
O.cuniculus mRNA for ATPase (alpha.. 
Chicken (Na+ + K+)-ATPase mRNA, co.. 
sodium pump alpha subunit [Ctenoce.. 
Mus musculus gastric H (+) -K(+) -ATP. . 
Homo sapiens (clone 1SW34) non-gas.. 
Dog H+,K+-ATPase mRNA, complete cds. 
Sheep mRNA for (Na+ and K+ ) ATPase.. 
Pig (H+ + K+)-ATPase mRNA, complet.. 
Rattus norvegicus Na,K-ATPase alph.. 
H(+)-K(+)-ATPase alpha-subunit [ra.. 
Rat Na+,K+-ATPase alpha isoform ca. . 
Rat sodium/potassium ATPase alpha-.. 
A.anguilla mRNA for sodium/potassi . . 
Rat mRNA for alpha subunit kidney-.. 
Torpedo californica mRNA for (Na+ . . 
Chicken NA,K-ATPase alpha-2-subuni . . 
Human Na,K-ATPase alpha-1 subunit .. 
Human mRNA for Na,K-ATPase alpha-s.. 
Human Na+,K+ ATPase alpha-subunit .. 
Canis familiaris Na, K-ATPase alph.. 
Pig NA+, K+-ATPase alpha subunit m. . 
Pig mRNA for (Na+, K+)-ATPase alph.. 
Rat Na+,K+-ATPase alpha(+) isoform.. 
C.commersoni mRNA for Na(+)/K(+) A.. 
A. franciscana mRNA for Na/K ATPase.. 
H. vulgaris Na, K-ATPase alpha subun.. 
Homo sapiens (clone 1SW11-1) non-g.. 
Na, K-ATPase alpha-1 subunit [dogs,.. 
Human gastric (H+ + K+)-ATPase gen.. 
Human gastric H, K-ATPase catalytic. 





Smallest 




Sum 


High Probabili 


Score 


P(N) 


1663 


4.1e-128 


1582 


2.2e-121 


1582 


2.2e-121 


1582 


2.2e-121 


1582 


2.2e-121 


1069 


l.le-78 


907 


3.1e-65 


898 


1.8e-64 


898 


1.8e-64 


889 


9.9e-64 


889 


9.9e-64 


880 


5.5e-63 


871 


3.1e-62 


853 


9.7e-61 


844 


5.5e-60 


835 


3.1e-59 


826 


1.7e-58 


826 


1.7e-58 


817 


9.6e-58 


458 


2.4e-56 


790 


1.7e-55 


772 


5.3e-54 


772 


5.3e-54 


763 


3.0e-53 


760 


5.3e-53 


736 


5.3e-51 


736 


5.3e-51 


727 


2.9e-50 


727 


2.9e-50 


667 


3.9e-49 


710 


7.6e-49 


709 


9.3e-49 


709 


9.3e-49 


709 


9.3e-49 


700 


5.2e-48 


691 


2.9e-47 


682 


1.6e-46 


674 


7.6e-46 


665 


9.0e-46 


646 


1.6e-43 


606 


3.4e-40 


551 


1.3e-35 


514 


1.5e-32 


318 


4.8e-24 


327 


5.5e-24 



Figure 4-3. BLAST search using sequence of 419 bp fragment of HKct : 



59 



P WGC3 , 



pWGC7 



pWGC9 



pWGCll 



RACE 



^ pWGC15 



pWGC53 



HKa* 



AUG... 



AUG.. 



HKa 2c 



UGA ...AAA 



^ pWGCM 



pWGC54 



Figure 4-4. Cloning of HKa 2a and HKoc 2c cDNAs. Individually cloned cDNAs are 
indicated by bars and the plasmid numbers are indicated. AUG and UGA indicate start 
and stop codons of the mRNA, respectively. Symbols: *, degenerated primers; RACE, 
RACE primer. All other primers were gene-specific. 















60 
attempts employed the classical technique of Frohman el al. (1988), which is analogous 

to the 3 'RACE process. In several attempts, this technique produced only short cDNA 
sequences which provided little new sequence information. Taking a new approach, the 
Marathon cDNA Amplification Kit by Clontech (Palo Alto, CA) was tried. This kit uses a 
ligation technology to add a known upstream sequence for PCR amplification, rather than 
the less efficient terminal transferase technology employed by Frohman el al. (1988). Also, 
the reverse transcription step was changed to a higher temperature to lessen the possibility 
of secondary structure blocking full extension by the RNA polymerase. With the new 
protocol, after some optimization, two different 5' ends were obtained when 5' RACE 
products were sequenced. 

Two rabbit renal HKct 2 cDNA sequences were found in this manner, having 4035 
bases in common at the 3' end but different 5' ends. The GenBank records of these two 
cDNAs are shown in Figure 4-5. A segment of the shared 3' portion of the sequences was 
identical to the 1456 bp sequence obtained by Fejes-Toth (1995) except for two single 
base mismatches. One mismatch was a transition of C->T at nucleotide 2927, which does 
not change the primary protein sequence and the other a transversion of G-+T at 
nucleotide 3259, in the 3' untranslated region. The full-length rabbit HKa 2 „ nucleotide 
sequence was 86% identical to human HKct: and 83% to rat, whereas identity of HKa 2a to 
rabbit HKa, was only 67%. The deduced amino acid sequence shared 87% identity with 
human HKa 2 , 87% identity with rat HKa : , but merely 64% with rabbit HKa,. Northern 
analysis using an HKa :a -specific probe (Table 2-1) is shown in Figure 4-6. 



A) 



61 



LOCUS 
DEFINITION 

ACCESSION 
NID 

KEYWORDS 
SOURCE 

ORGANISM 



REFERENCE 
AUTHORS 
TITLE 

JOURNAL 
REFERENCE 
AUTHORS 
TITLE 
JOURNAL 

FEATURES 

source 



CDS 



AF023128 4079 bp mRNA MAM 13-OCT-1997 

Oryctolagus cuniculus H+,K+-ATPase alpha 2a subunit mRNA, 

complete cds. 

AF023128 

g2511766 

Oryctolagus cuniculus. 

Oryctolagus cuniculus 

Eukaryotae; Metazoa; Chordata; Vertebrata; Mammalia; 

Eutheria; Lagomorpha; Leporidae; Oryctolagus. 

1 (bases 1 to 4079) 

Campbell, W. G. , Weiner,I.D., Wingo,C.S. and Cain,B.D. 
H,K-ATPase in the RCCT-28A rabbit cortical collecting duct 
cell line 
Unpublished 

2 (bases 1 to 4079) 

Campbell, W. G. , Wingo,C.S. and Cain,B.D. 

Direct Submission 

Submitted (08-SEP-1997 ) Biochemistry, University of Florida, 

JHMHC 100245, Gainesville, FL 32610, USA 

Location /Qualifiers 

1. .4079 

/organ ism= "Oryctolagus cuniculus" 

/strain="New Zealand White" 

/db_xref="taxon: 9986" 

39. .3140 

/note="P-type ATPase" 

/codon_start=l 

/product="H+,K+-ATPase alpha 2a subunit" 

/db_xref="PID:g2 511767" 

/translation^' MRQRKLE I YSVELHAATD I KKKEGRDGKKDNDLELKRNQQKEEL 
KKELDLDDHKLSNKELETKYGTDIIRGLSSTRAAELLAQNGPNALTPPKQTPEIIKFL 
KQMVGGFSILLWVGAVLCWIAFGIQYVSNPSASLDRVYLGTVLAVVVILTGIFAYYQE 
AKSTNIMASFCKMIPQQAVVIRDSEKKVIPAEQLVVGDIVEIKGGDQIPADIRLLSAQ 
GCKVDNSSLTGESEPQSRSSGFTHENPLETKNITFYSTTCLEGTATGMVINTGDRTII 
GRIASLASGVGNEKTPIAIEIEHFVHIVAGVAVSVGILFFIIAVCMKYHVLDAIIFLI 
AI IVANVPEGLLATVTVALSLTAKRVAKKNCLVKNLEAVETLGSTSI ICSDKTGTLTQ 
NRMTVAHLWFDNQIFVADTSEDNLNQGFDQSSGTWTSLSKIIALCNRAEFKPGEESVP 
IMKRVVVGDASETALLKFSEVILGDVMEIRKRNHKVVEIPFNSTNKFQLSIHQTEDPN 
DKRFLLVMKGAPERILEKCSTIMINGKEQPLDKSMAQAFHTAYMELGGLGERVLGFCH 
FYLPADEFPETYSFDSESMNFPTSNLCFVGLLSMIDPPRSTVPDAVTKCRSAGIKVIM 
VTGDHP I TAKAI AKSVG I I SANSETVED I AKRCN I AVEQVNKRDAKAAVVTGMELKDM 
SPEQLDELLANYPEIVFARTSPQQKLIIVEGCQRQDAVVAVTGDGVNDSPALKKADIG 
VAMGITGSDAAKNAADMILLDDNFSSIVTGVEEGRLIFDNLKKTIAYTLTKNIAELCP 
FLIYIILGLPLPIGTITLLFIDLGTDIIPSIALAYEKAESDIMNRKPRHKKKDRLVNQ 



Figure 4-5. GenBank accession records for rabbit HKa : sequences. A) Genbank record 
for rabbit HKa 2i , B) Genbank record for rabbit HKoc 2e . 



62 

QLAVYSYLHIGLMQALGAFLVYFTVYAQQGFRPTSLFHLRIAWDSDHLNDLEDNYGQE 
WTSYQRQYLEWTGYTAFFVGIMVQQIADLIIRKTRKNSIFKQGLFRNKVIWVGIASQI 
IVALLLSYGLGSITALNFTMLKAQYWFVAVPHAILIWVYDEMRKLFIRLYPGSWWDKN 
MYY" 
BASE COUNT 1037 a 1073 c 1024 g 945 t 
ORIGIN 

1 gccccctgcc cgccgacccg cggcgcctcc agcgcgacat gcgccagaga aagctggaaa 
61 tttactccgt ggagctccat gcagctacag atatcaagaa gaaggagggg cgagatggca 
121 agaaagacaa tgacttggaa ctcaaaagga atcagcagaa agaggagctt aagaaagaac 
181 ttgatctgga tgaccacaaa ctcagcaata aggagctgga aacgaaatat ggcacagaca 
241 tcattcgggg tctctccagc accagagctg ctgagctcct ggcacagaac ggacccaacg 
301 ccctcacccc tcccaaacag accccagaga tcatcaagtt cctcaagcag atggtgggcg 
361 gcttttccat ccttctgtgg gtaggagctg tcctgtgttg gatcgcattt gggattcagt 
421 atgtcagcaa tccatctgcc tccctggaca gagtgtacct gggcactgta cttgccgtgg 
481 ttgtcatttt aacaggaatc tttgcctatt accaagaggc aaaaagcacc aacatcatgg 
541 ccagcttctg caagatgatc ccccagcaag ctgttgtcat ccgtgactcg gagaaaaagg 
601 ttatccctgc agagcagctg gtggtggggg acatcgtgga gattaaagga ggtgaccaga 
661 ttcctgccga catcaggctg ctgtctgccc aggggtgtaa ggtggataac tcatctctta 
721 ctggagagtc tgagccccag tcccgctcaa gtgggttcac ccacgaaaac cccctggaaa 
781 caaagaacat cactttctac tccacgacct gcctggaagg cacggcaact ggcatggtca 
841 tcaacacggg tgaccggacc atcattggcc gcattgcctc cttggcttca ggcgtcggga 
901 atgagaagac gcccattgcc attgagatcg aacattttgt gcacattgtg gcaggagtgg 
961 ccgtctccgt cggcatcctg ttcttcatca tcgcagtgtg catgaagtac cacgtcctgg 
1021 acgccatcat cttcctcatt gccatcattg tggccaacgt gcctgaaggc ctcctggcca 
1081 ctgtcactgt ggccctgtcg ctcacagcca aacgggtggc caagaagaac tgcctggtga 
1141 agaacttgga ggcagtggag accctcggct ccacctccat catctgctct gacaagactg 
1201 ggactctgac gcagaacagg atgaccgtgg cccatctgtg gtttgacaat cagatcttcg 
1261 tggccgacac gagtgaagac aatttaaacc aaggctttga ccaaagctct ggaacctgga 
1321 cctccttgtc caagataata gcattgtgta accgagctga gttcaagcca ggagaggaga 
1381 gtgtccccat catgaagaga gtcgtggttg gagatgcttc agaaactgct cttctgaaat 
1441 tctcagaagt cattttgggt gacgtgatgg aaattagaaa aagaaaccac aaagtagtcg 
1501 aaatcccttt taactcaacc aacaaatttc agctctccat acaccagacg gaagatccca 
1561 atgacaagcg cttcctgctg gtgatgaagg gggcccccga gcggatccta gagaagtgca 
1621 gcaccatcat gatcaacggc aaggagcagc cactggacaa gagcatggcc caggccttcc 
1681 acacggccta catggagctg ggcggcctgg gcgagcgcgt gctgggtttc tgccatttct 
1741 acctgccagc agatgagttt ccagagacct actcatttga ctcagaatcc atgaacttcc 
1801 ccacctccaa cttatgtttt gtggggctct tatcaatgat tgatcctcct cgatccactg 
1861 tcccagatgc agtcaccaaa tgccggagtg caggaatcaa ggttatcatg gttacaggtg 
1921 atcatcccat cacagccaaa gccattgcca agagtgtagg gatcatttca gccaacagtg 
1981 agacagtgga agacattgca aaacgctgca acatcgccgt ggagcaggtt aacaaacggg 
2041 atgccaaggc cgccgtggtg accggcatgg agctgaagga catgagccca gaacagctgg 
2101 atgagctctt agccaactac ccggaaatcg tgtttgcacg gacgtccccc cagcaaaagc 
2161 tgatcatcgt ggagggctgt cagaggcagg acgcagttgt ggccgtgacg ggggacggag 
2221 tgaatgactc ccccgctcta aagaaggccg acattggcgt tgccatgggg ataacgggtt 
2281 ctgacgcggc caagaacgca gccgacatga tcctgctgga tgacaacttc tcctctatcg 
2341 tcacaggggt ggaggaaggc cgcttgatat ttgacaacct aaagaagacc atcgcttaca 
2401 ccctgaccaa gaacattgcc gagctctgcc cctttttgat ttacatcatt ctcgggctgc 
2461 ccctgcccat tggcaccatc accctcctgt tcatcgactt gggcacagac ataatcccct 
2521 ccattgcctt ggcgtatgag aaagcagaaa gtgacattat gaacaggaag cctcggcaca 
2581 agaaaaagga cagactggtg aaccagcagc ttgctgtata ctcgtacctg cacattggcc 
2641 tcatgcaagc cctgggagct ttcctggtgt acttcactgt gtacgcacag cagggctttc 
2701 ggccgacctc actgtttcac ctgcggatag cgtgggacag cgaccacctg aacgacttgg 

Figure 4-5~continued 



63 

2761 aagacaacta tggacaggaa tggacgagtt atcagaggca atacctggaa tggacaggct 
2821 acacggcttt ctttgttggc atcatggtcc agcaaatagc agatctgatc atcaggaaga 
2881 cccgcaagaa ctccatcttc aagcaggggc tcttcagaaa taaagttatc tgggtgggga 
2941 tcgcctccca gatcatcgtc gccctgctcc tctcttacgg gctcggcagt atcacagccc 
3001 taaatttcac catgctcaag gctcagtact ggtttgtggc cgtaccccac gccatcctga 
3061 tctgggtata cgatgaaatg cggaaactct tcatcaggct ctaccccgga agctggtggg 
3121 ataagaacat gtattactga gaccaggtct gtctctgagt ctcccagcgg cacctgcctg 
3181 gtggtcttcg gcaagacctc tgtgtagtgt ggatgttgcc aagctccact cgggaggaga 
3241 ctctcatcta gaacacagtg gtgaagcttc ttactgatct gttgtacttc aaagctgaga 
3301 ttcagctgtt tgtatatgat tttcatctct atctccatct ccttacctta aaagatgtgg 
3361 atgtcaaggt catggtgtag ggaaggatgt gtttatctgt atatgaagct cactgatgtc 
3421 acacagactt gtgtaaccca ggtggctgct ggagtctgcc ataagttgag ctagaattgc 
3481 tcagatctcc ttccacaccc tgtcaaaggc ccggtgagct ccataggatt tctgtgaatc 
3541 cccctgaaac ataacttttg gggtttgctt tgctcagctg agggtgtgag ttggaagtgt 
3601 ggcagcagga gcacctcaga acagcaaaga cagcccccgt tttgactccc agacactttg 
3661 ttgctgtgat gggttcctgg ccatgcggcc ccagtccgcc ttctcacagc actccaccac 
3721 ctgttcctgc aaagctgacc tccaagtcca ttccacaaac cttaactcaa acattcgtgg 
3781 acccaaaggg gctgtcactg actgggactc ggcctctccg gaaagccact gtggtttaga 
3841 tagcactatt tatttcttgt agataggctg ccaagcactc tccagcagcc attttatgtc 
3901 aatcacattt ttgtaactta gatatatttg tgtgggacac gaaacacata catccatgtt 
3961 gacaggtttt tttttttaaa taaaagatgt ttttaagtaa aatgttttat gaaacaaaat 
4021 ctaattgtga tgttttactt aattcaagtt tttccagagg caggcacgga aaataccaa 
// 



Figure 4-5-continued 



B) 



64 



LOCUS 
16-APR-1998 

DEFINITION 

ACCESSION 
NID 

KEYWORDS 
SOURCE 

ORGANISM 



AF023129 



4422 bp 



mRNA 



MAM 



REFERENCE 
AUTHORS 
TITLE 

JOURNAL 
REFERENCE 
AUTHORS 
TITLE 
JOURNAL 



FEATURES 

source 



CDS 



Oryctolagus cuniculus H+,K+-ATPase alpha 2c subunit mRNA, 

complete cds. 

AF023129 

g2511768 

Oryctolagus cuniculus. 

Oryctolagus cuniculus 

Eukaryotae; Metazoa; Chordata; Vertebrata; Mammalia; 

Eutheria; 

Lagomorpha; Leporidae; Oryctolagus. 

1 (bases 1 to 4422) 

Campbell, W. G. , Weiner,I.D., Wingo,C.S. and Cain,B.D. 
H,K-ATPase in the RCCT-28A rabbit cortical collecting duct 
cell line 
Unpublished 

2 (bases 1 to 4422) 

Campbell, W.G. , Wingo,C.S. and Cain,B.D. 

Direct Submission 

Submitted (08-SEP-1997 ) Biochemistry, University of Florida, 

JHMHC 

100245, Gainesville, FL 32610, USA 

Location/Qualifiers 

1. .4422 

/ organ ism= "Oryctolagus cuniculus" 

/strain="New Zealand White" 

/db_xref="taxon:9986" 

199. .3483 

/note="P-type ATPase" 
/codon_start=l 

/product="H+,K+-ATPase alpha 2c subunit" 
/db_xref="PID:g2511769" 

/translation="MAGGAHRADRATGEERKEGGGRWRAPHSPSPPGPRGCPVPLKAA 
AQSLCRKPTWGRYCTLLLFQRKLEIYSVELHAATDIKKKEGRDGKKDNDLELKRNQQK 

EELKKELDLDDHKLSNKELETKYGTDIIRGLSSTRAAELLAQNGPNALTPPKQTPEII 
KFLKQMVGGFSILLWVGAVLCWIAFGIQYVSNPSASLDRVYLGTVLAWVILTGIFAY 
YQEAKSTNIMASFCKMIPQQAVVIRDSEKKVIPAEQLVVGDIVEIKGGDQIPADIRLL 
SAQGCKVDNSSLTGESEPQSRSSGFTHENPLETKNITFYSTTCLEGTATGMVINTGDR 
TIIGRIASLASGVGNEKTPIAIEIEHFVHIVAGVAVSVGILFFIIAVCMKYHVLDAII 
FLIAIIVANVPEGLLATVTVALSLTAKRVAKKNCLVKNLEAVETLGSTSIICSDKTGT 
LTQNRMTVAHLWFDNQIFVADTSEDNLNQGFDQSSGTWTSLSKIIALCNRAEFKPGEE 
S VP IMKRVVVGDASETALLKFSE V I LGD VME I RKRNHKVVE I PFNSTNKFQLS I HQTE 
DPNDKRFLLVMKGAPERILEKCSTIMINGKEQPLDKSMAQAFHTAYMELGGLGERVLG 
FCHFYLPADEFPETYSFDSESMNFPTSNLCFVGLLSMIDPPRSTVPDAVTKCRSAGIK 
VIMVTGDHPITAKAIAKSVGIISANSETVEDIAKRCNIAVEQVNKRDAKAAWTGMEL 
KDMSPEQLDELLANYPEIVFARTSPQQKLIIVEGCQRQDAVVAVTGDGVNDSPALKKA 
DIGVAMGITGSDAAKNAADMILLDDNFSSIVTGVEEGRLIFDNLKKTIAYTLTKNIAE 



Figure 4-5~continued 



65 

LCPFLIYIILGLPLPIGTITLLFIDLGTDIIPSIALAYEKAESDIMNRKPRHKKKDRL 
VNQQLAVYSYLHIGLMQALGAFLVYFTVYAQQGFRPTSLFHLRIAWDSDHLNDLEDNY 
GQEWTSYQRQYLEWTGYTAFFVGIMVQQIADLIIRKTRKNSIFKQGLFRNKVIWVGIA 
SQIIVALLLSYGLGSITALNFTMLKAQYWFVAVPHAILIWVYDEMRKLFIRLYPGSWW 
DKNMYY" 
BASE COUNT 1103 a 1174 c 1136 g 1009 t 
ORIGIN 

1 ctccgccctc gcacctgcgg gctcggattc ggagaaaagt gctagactgg agctacacgt 
61 atgcgtagcg gtctggaaaa tgccccaggc tcgggtctga ggggcccaag tctatgcacc 
121 gctggtgtga ccccgcaggg caaccccgcg gttaacttct ctcctgccca cccctagagg 
181 tgtcttcctg ggaagacgat ggcaggcggt gcccaccgag ccgaccgtgc aacaggggaa 
241 gagaggaagg agggaggtgg gaggtggcgc gctccccaca gcccttcccc tcctggcccg 
301 cgagggtgtc cggtcccact caaggcagct gcgcagagcc tgtgcagaaa acccacctgg 
361 ggccggtatt gcactctgct tctctttcag agaaagctgg aaatttactc cgtggagctc 
421 catgcagcta cagatatcaa gaagaaggag gggcgagatg gcaagaaaga caatgacttg 
481 gaactcaaaa ggaatcagca gaaagaggag cttaagaaag aacttgatct ggatgaccac 
541 aaactcagca ataaggagct ggaaacgaaa tatggcacag acatcattcg gggtctctcc 
601 agcaccagag ctgctgagct cctggcacag aacggaccca acgccctcac ccctcccaaa 
661 cagaccccag agatcatcaa gttcctcaag cagatggtgg gcggcttttc catccttctg 
721 tgggtaggag ctgtcctgtg ttggatcgca tttgggattc agtatgtcag caatccatct 
781 gcctccctgg acagagtgta cctgggcact gtacttgccg tggttgtcat tttaacagga 
841 atctttgcct attaccaaga ggcaaaaagc accaacatca tggccagctt ctgcaagatg 
901 atcccccagc aagctgttgt catccgtgac tcggagaaaa aggttatccc tgcagagcag 
961 ctggtggtgg gggacatcgt ggagattaaa ggaggtgacc agattcctgc cgacatcagg 
1021 ctgctgtctg cccaggggtg taaggtggat aactcatctc ttactggaga gtctgagccc 
1081 cagtcccgct caagtgggtt cacccacgaa aaccccctgg aaacaaagaa catcactttc 
1141 tactccacga cctgcctgga aggcacggca actggcatgg tcatcaacac gggtgaccgg 
1201 accatcattg gccgcattgc ctccttggct tcaggcgtcg ggaatgagaa gacgcccatt 
1261 gccattgaga tcgaacattt tgtgcacatt gtggcaggag tggccgtctc cgtcggcatc 
1321 ctgttcttca tcatcgcagt gtgcatgaag taccacgtcc tggacgccat catcttcctc 
1381 attgccatca ttgtggccaa cgtgcctgaa ggcctcctgg ccactgtcac tgtggccctg 
1441 tcgctcacag ccaaacgggt ggccaagaag aactgcctgg tgaagaactt ggaggcagtg 
1501 gagaccctcg gctccacctc catcatctgc tctgacaaga ctgggactct gacgcagaac 
1561 aggatgaccg tggcccatct gtggtttgac aatcagatct tcgtggccga cacgagtgaa 
1621 gacaatttaa accaaggctt tgaccaaagc tctggaacct ggacctcctt gtccaagata 
1681 atagcattgt gtaaccgagc tgagttcaag ccaggagagg agagtgtccc catcatgaag 
1741 agagtcgtgg ttggagatgc ttcagaaact gctcttctga aattctcaga agtcattttg 
1801 ggtgacgtga tggaaattag aaaaagaaac cacaaagtag tcgaaatccc ttttaactca 
1861 accaacaaat ttcagctctc catacaccag acggaagatc ccaatgacaa gcgcttcctg 
1921 ctggtgatga agggggcccc cgagcggatc ctagagaagt gcagcaccat catgatcaac 
1981 ggcaaggagc agccactgga caagagcatg gcccaggcct tccacacggc ctacatggag 
2041 ctgggcggcc tgggcgagcg cgtgctgggt ttctgccatt tctacctgcc agcagatgag 
2101 tttccagaga cctactcatt tgactcagaa tccatgaact tccccacctc caacttatgt 
2161 tttgtggggc tcttatcaat gattgatcct cctcgatcca ctgtcccaga tgcagtcacc 
2221 aaatgccgga gtgcaggaat caaggttatc atggttacag gtgatcatcc catcacagcc 
2281 aaagccattg ccaagagtgt agggatcatt tcagccaaca gtgagacagt ggaagacatt 
2341 gcaaaacgct gcaacatcgc cgtggagcag gttaacaaac gggatgccaa ggccgccgtg 
2401 gtgaccggca tggagctgaa ggacatgagc ccagaacagc tggatgagct cttagccaac 
2461 tacccggaaa tcgtgtttgc acggacgtcc ccccagcaaa agctgatcat cgtggagggc 
2521 tgtcagaggc aggacgcagt tgtggccgtg acgggggacg gagtgaatga ctcccccgct 
2581 ctaaagaagg ccgacattgg cgttgccatg gggataacgg gttctgacgc ggccaagaac 



Figure 4-5~continued 



66 

2641 gcagccgaca tgatcctgct ggatgacaac ttctcctcta tcgtcacagg ggtggaggaa 
2701 ggccgcttga tatttgacaa cctaaagaag accatcgctt acaccctgac caagaacatt 
2761 gccgagctct gccccttttt gatttacatc attctcgggc tgcccctgcc cattggcacc 
2821 atcaccctcc tgttcatcga cttgggcaca gacataatcc cctccattgc cttggcgtat 
2881 gagaaagcag aaagtgacat tatgaacagg aagcctcggc acaagaaaaa ggacagactg 
2941 gtgaaccagc agcttgctgt atactcgtac ctgcacattg gcctcatgca agccctggga 
3001 gctttcctgg tgtacttcac tgtgtacgca cagcagggct ttcggccgac ctcactgttt 
3061 cacctgcgga tagcgtggga cagcgaccac ctgaacgact tggaagacaa ctatggacag 
3121 gaatggacga gttatcagag gcaatacctg gaatggacag gctacacggc tttctttgtt 
3181 ggcatcatgg tccagcaaat agcagatctg atcatcagga agacccgcaa gaactccatc 
3241 ttcaagcagg ggctcttcag aaataaagtt atctgggtgg ggatcgcctc ccagatcatc 
3301 gtcgccctgc tcctctctta cgggctcggc agtatcacag ccctaaattt caccatgctc 
3361 aaggctcagt actggtttgt ggccgtaccc cacgccatcc tgatctgggt atacgatgaa 
3421 atgcggaaac tcttcatcag gctctacccc ggaagctggt gggataagaa catgtattac 
3481 tgagaccagg tctgtctctg agtctcccag cggcacctgc ctggtggtct tcggcaagac 
3541 ctctgtgtag tgtggatgtt gccaagctcc actcgggagg agactctcat ctagaacaca 
3601 gtggtgaagc ttcttactga tctgttgtac ttcaaagctg agattcagct gtttgtatat 
3661 gattttcatc tctatctcca tctccttacc ttaaaagatg tggatgtcaa ggtcatggtg 
3721 tagggaagga tgtgtttatc tgtatatgaa gctcactgat gtcacacaga cttgtgtaac 
3781 ccaggtggct gctggagtct gccataagtt gagctagaat tgctcagatc tccttccaca 
3841 ccctgtcaaa ggcccggtga gctccatagg atttctgtga atccccctga aacataactt 
3901 ttggggtttg ctttgctcag ctgagggtgt gagttggaag tgtggcagca ggagcacctc 
3961 agaacagcaa agacagcccc cgttttgact cccagacact ttgttgctgt gatgggttcc 
4021 tggccatgcg gccccagtcc gccttctcac agcactccac cacctgttcc tgcaaagctg 
4081 acctccaagt ccattccaca aaccttaact caaacattcg tggacccaaa ggggctgtca 
4141 ctgactggga ctcggcctct ccggaaagcc actgtggttt agatagcact atttatttct 
4201 tgtagatagg ctgccaagca ctctccagca gccattttat gtcaatcaca tttttgtaac 
4261 ttagatatat ttgtgtggga cacgaaacac atacatccat gttgacaggt tttttttttt 
4321 aaataaaaga tgtttttaag taaaatgttt tatgaaacaa aatctaattg tgatgtttta 
4381 cttaattcaa gtttttccag aggcaggcac ggaaaatacc aa 
// 



Figure 4-5-continued 



67 




a 2a •**' -4.1 kb 



- 



GAP3DH • m - 1.3 kb 

Distal Renal 
Colon Cortex 



Figure 4-6. Northern analysis showing presence of HKoc^ in distal colon and renal 
cortex. GAP3DH was used to show the condition of the RNA samples. 






68 
To determine the relationship between the rabbit HKot: mRNAs and other P-type 

ATPases, phylogenetic analysis was necessary. Several programs and algorithms were 
sampled, all giving the same general pattern of sequence relationships. Programs employed 
include CLUSTALW (Thompson el al., 1994), DISTANCES (Genetics Computer Group, 
1997), and PAUP (Genetics Computer Group, 1997). All three programs use the distance 
algorithm, and the maximum parsimony algorithm of PAUP was also used. In addition to 
distance and maximum parsimony analyses, a third popular algorithm to assay relatedness 
of sequences is used, the maximum likelihood analysis. Distance analysis generates a tree 
showing relatedness of sequences by simply counting 

dissimilarities in aligned sequences as a test of their homology. Maximum parsimony 
considers only positive information in making a comparison. For instance, in an alignment 
of four sequences, a position that had one each of A, C, G, and T would not be included in 
the analysis because that position would not give information about any pair of the 
sequences being related. A position that had two As and two Cs would be counted 
because it would show the grouping of two pairs. Distance analysis would include both 
positions in the analysis. Maximum likelihood takes into account the tendency for a given 
type of mutation to occur. In two sequences that have a point mutation, that point 
mutation was more probably created by a "likely" change than a change that has less 
tendency to occur. Maximum likelihod would measure two sequences as more related if 
their alignment shows a "likely" mutation. If the mutation had less tendency, it may 
indicate a more distant relationship between sequences arrived at only by multiple changes 



69 
or higher pressure of selection. The maximum likelihood analysis requires information that 

is not readily available for rabbit, and was not attempted. A representative phylogram 
arrived at by distance analysis by the CLUSTALW program is shown in Figure 4-7. 
The 5' end of the second rabbit sequence (HKa; c ) differed dramatically from the 
published rat HKa 2 „ (Crowson and Shull, 1992) and human HKct 2a (Grishin et cif., 1994) 
sequences. The HKot 2i; 5' untranslated region bore no homology to the comparable 
segments from the HKa 2 „ cDNAs from human or rat, or to any GenBank sequence. It 
was clear from the beginning that this second 5' end might represent an alternative splicing 
product; the sequence homology diverges from the human HKoc 2 sequence at a point 
known in the human ATP1 AL1 gene to be a splice junction (nucleotide 177 of ATP1AL1) 
(Sverdlove/a/., 1996). 

Alternative Splicing of H.K-ATPase a Subunits in the Kidney 

When a sequence for the 5' end of the rat HKa 2 gene was published (Kone and 
Higham, 1998), we wanted to compare the sequence at the 5' end of the rabbit gene. In 
order to determine the exon arrangement at the 5' end of the HKoc 2 gene, rabbit genomic 
DNA was amplified by PCR. The antisense primer was selected within the region of 
sequence that is common to HKa 2i , and HKa 2t . Because it was unknown which of the two 
cDNA 5' ends was located more 5' in the gene, sense primers that anneal to sequence 
within each were selected. The oligonucleotide primer specific to HKa 2: , gave the larger 
amplified product, and its sequence is shown in Figure 4-8A. The exon specific to HKa 2a 
splices to the common core sequence at the splice site shown. The exon specific to HKa 2c 



70 



rabbit HKal 
human HKal 



rat HKal 



toad HKa3 



rat HKa2 

rabbit HKa2 




rat NaKa2 



human NaKa2 



rat NaKal 

human NaKal 
4aKal 



human HKa2 



Figure 4-7. Distance analysis of selected HKcc and NaKa subunit codin». 



A) 



T HKota. transcription start 
1 GCCCCCTGCC CGCCGACCCG CGGCGCCTCC AGCGCGACAT GCGCCAGgtg 

M R Q 



71 



B) 



51 tgtgaggaag tgacgcggtg cggactggcg agaagtgcgg gaaagggtga 



101 agggctccgt ccgggggtct ttactctgca accctgttcc agccgccgag 



151 cacccgtgtg tcactcggga actggctggg t^aagaggtc aatccagaca 



M a 



HKa JC transcription 
201 cgcggggaag gagttccagg ggtcctgggc cagCTCCGCC CTCGCACCTG 

start 
251 CGGGCTCGGA TTCGGAGAAA AGTGCTAGAC TGGAGCTACA CGTATGCGTA 



301 GCGGTCTGGA AAATGCCCCA GGCTCGGGTC TGAGGGGCCC AAGTCTATGC 



351 ACCGCTGGTG TGACCCCGCA GGGCAACCCC GCGGTTAACT TCTCTCCTGC 



4 01 CCACCCCTAG AGGTGTCTTC CTGGGAAGAC GATGGCAGGC GGTGCCCACC 

MAG G A H R 



4 51 GAGCCGACCG TGCAACAGGG GAAGAGAGGA AGGAGGGAGG TGGGAGGTGG 
ADR ATC GEER KEG GGW 



501 CGCGCTCCCC ACAGCCCTTC CCCTCCTGGC CCTCGAGGGT GTCCGGTCCC 
RAPH SPS PPG PRGC PVP 

551 ACTCAAGGCA GCTGCGCAGA GJCTGTGCAG AAAACCCACC TGGGGCCGGT 
LKA AAQS LCR KPT WGRY 

HKa,. splice site 
601 ATTGCACTCT GCTTCTCTTT CAGAGAAAGC TGGAAATTTA CTCCGTGGAG 
CTL LLF QRKL EIY SVE 



651 ... remainder of sequence omitted for clarity 



HKa 



2a 



HKa 



2c 



...ATG.71 

pntron 


...ATG... 


...atg... COMMON 



splicing 



Figure 4-8. Rabbit HKcc 2 gene sequence at the 5* end. A) HKoc 2 gene sequence and 
deduced amino acid sequences are shown. Intron sequence is in lower case type. The 
amino acid sequence that is shared by HKct 2a and HKoc 2c is in boldface type. B) Pattern of 
alternative splicing at the 5' ends of the HKcc 2 transcripts. 



72 
is continuous with the core sequence and lies within the first intron of the HKa 2a 

pre-mRNA. This general intron/exon structure is the same as that reported for rat HKa 2a 

and HKa 2b (Kone and Higham, 1998). 

Expression of H,K-ATPase a Subunits in the Kidney 

The start codon of HKa 2 „ was omitted in the HKa 2c sequence. Instead, a probable 
start codon was located upstream in frame with the HKa 2a open reading frame. Thus, the 
deduced amino acid sequence encoded a protein 61 amino acids longer at the amino 
terminal end than the rabbit HKa 2a sequence. The deduced amino acid sequence of the 
HKa 2a and HKoc 2c proteins are shown in Figure 4-8A, indicating translational start sites. 
Although the HKa 2t cDNA indicated a continuous open reading frame including the 
amino-terminal extension, the possibility remained that translation might be initiated at the 
ATG codon homologous to that reported for the rat (Kone and Higham, 1998). 
Therefore, to determine whether the upstream ATG served as a translational start site, 
antipeptide antibodies were generated for peptides corresponding to amino acids 13-25 
and 79-98 of the HKa 2c subunit. The former (antibody LLC27) was HKct 2c -specific, while 
the latter (antibody LLC25) recognized a segment common to both HKa 2a and HKa 2c 
subunits. Western analyses of rabbit kidney tissues and RCCT-28A cells using antibody 
LLC25 (anti-HKa 2 common) revealed a doublet migrating at an apparent molecular mass 
of approximately 90KDa (figure 4-9A). Experiments using antibody LLC27 (anti-HKa 2c ) 
indicated a single band with a migration comparable to the upper band of the doublet 
(figure 4-9B). Both antibodies appear to recognize the same protein providing strong 



73 



A) 



B) 



anti-HKot 2 common anti-HKa 



2c 



a 2c 

Ot2a 



a 2c - 



. ._.-. <1 



O CD 

x 




-66.2 



Figure 4-9. Western analysis showing presence of HKa 2a and HKa 2c protein in renal 
cortex. A) Detected with the anti-HKa 2 common antibody LLC25. B) Detected with the 
anti-HKcc 2c -specific antibody LLC27. A and B show separate lanes from the same 
SDS-PAGE gel. Membrane was cut after electrotransfer and re-aligned after 
autoradiography. 



74 
evidence that the HKct2 C subunit containing the amino-terminal extension was indeed 

present in both rabbit renal tissue and RCCT-28A cells. 

To test the hypothesis that enzyme localization in the kidney or in the cell is changed 
as a result of the alternative splicing of the mRNA, immunohistochemistry experiments 
were performed. Using our antibodies, Dr. Jill Verlander and Ms. Robin Moudy 
conducted immunohistochemistry experiments. Neither antibody we had made to the 
anti-HKa 2 common region (LLC24 or LLC25) worked well for immunohistochemistry. 
However LLC22 (anti-HKai) and LLC26 (anti-HKooc) gave satisfactory results. A 
representative section photographed in Dr. Verlander' s laboratory is shown in Figure 
4-10. There is no visible reactivity except in the apical membranes of some cells of 
connecting segment and collecting duct. A majority of the cells in which labelling is 
observed bulge out into the lumen, a hallmark of the intercalated cell. Fewer cells are 
labelled in connecting segment compared to more distally in the collecting duct. This is in 
accordance with the normal distribution of acid-secreting intercalated cells, fewer in 
connecting segment than farther down in cortical collecting duct. There appears to be 
some labelling of cell types other than intercalated cells, including principal cells. Labelling 
continues to be visible in more distal sections of collecting duct than one would expect 
based on distribution of intercalated cells, but individual rabbits may vary widely in this 
distribution. Therefore, the few observations made do not permit a certainty that HKa 2c 
subunit protein is expressed more distally in cell types not normally thought to be 
associated with acid secretion. The lack of basolateral labelling implies that there is not a 
cell polarity change in HKa 2c protein compared to HKa 2a . 



75 




m € : : 



Figure 4-10. Immunohistochemistry by Dr. Jill Verlander and Ms. Robin Moudy. 
Anti-HKa 2c antibody reacts with the apical surfaces in some cells in rabbit renal 
collecting duct. The section shown is from the outer stripe of outer medulla. 



76 



Discussion 

We have generated complete cDNAs for both the rabbit renal HKa-, and the novel 
HKa 2c subunits. They were more closely related to other HKa 2 nucleotide sequences than 
to HKa, or to Na,K-ATPases. The exon structure at the 5' end was found and compared 
to the exon structure found at the 5' end of rat (Kone and Higham, 1998). Proteins 
corresponding to both HKa : isoforms were detected by immunoblot analysis, indicating 
that the novel HKa 2c has the predicted amino-terminal extension. 

The HKa 2 cDNAs reported here generated using rabbit renal cortex RNA as template 
have high homology to previously known HKa : sequences from human skin axilla and the 
rat distal colon (Grishin et al., 1994; Crowson and Shull, 1992). The level of homology is 
relatively low compared to the homology typically found when comparing HKa, or NaKa 
subunits across mammalian species. However, it is much higher than the homology 
between the rat HKa, and HKa : cDNAs (Crowson and Shull, 1992) or among the 
different Na,K-ATPase isoforms within a species. The HKa : isoforms appear to have 
undergone somewhat greater evolutionary divergence than HKa, or the Na,K-ATPase 
catalytic isoforms. Phylogenetic analysis provides a clearer picture of the relationships 
between these P-type ATPases. In a phylogram (figure 4-7), the HKc* : subunit isoforms 
cluster together compared to the other P-type ATPases. The HKa : subunit nucleotide 
sequences all represent H,K-ATPases that are expressed at high level in colon, at lower 
level in other tissues including the kidney, and not at all in stomach. At least in rat and 



77 
rabbit, an alternatively spliced variant has been shown to be transcriptionally competent, 

and the pattern of introns and exons that give rise to this alternative splicing are strikingly 

similar. It would be highly interesting to attempt to detect a similar transcript in human. 

Based on tissue distribution, phylogenetic analysis, and the similarity of alternatively 

spliced transcripts, it continues to be a valid premise that the HKcc: subunits known for 

rat, human, and rabbit form an orthologous group. 

Like the rat, the rabbit appears to have alternatively spliced transcripts of HKa 2 in the 
kidney. The organization of the rat alternatively spliced cDNA (Kone and Higham, 1998) 
omitted the exon containing the start codon of the sequence previously reported for rat 
distal colon (Crowson and Shull, 1992), giving rise to a protein truncated by 108 amino 
acids at the ainino-terminal end. The rabbit HKa 2c sequence also omits the start codon 
present in the HKa-, sequence. However, an upstream 5' ATG codon lies in the same 
uninterrupted reading frame as the coding sequence for HKa 2 . Initiation of translation at 
this position yields a subunit having an extended ammo-terminus 6 1 amino acids longer 
than the canonical HKa 2a protein. Western analysis demonstrated that a protein having 
this extension is present in rabbit kidney. An antibody designed to detect the common 
core region of HKa 2 was reactive to two proteins very close in apparent mass, whereas an 
antibody designed to detect the amino-terminal extension was reactive to a single species. 

The extended portion of the HKa 2b -encoded protein contains a casein kinase II 
phosphorylation motif at thr-12, and a cAMP-dependent protein kinase phosphorylation 
motif at thr-53. These sites impart a potential for regulation of HKa 2c distinct from HKa 2a . 
There are no apparent glycosylation sites or signal sequences. The HKa 2c extension is 



78 
hydrophilic in nature and lacks any conspicuous membrane-spanning domains. Because the 

N-terminus of HKa, was found to be cytosolic (Smolka et a/., 1992), the elongation can 
be expected to have a cytosolic location. Figure 1-1 shows the putative location of the 
amino-terminal extension. Chou-Fasman calculations predict this segment to be 
predominantly alpha- helical with a turn structure in a region containing seven prolines 
between amino acids 26 through 40. A similarly proline-rich hinge region is found in band 
3 protein, and an ankyrin-binding site has been localized to that area of band 3 (Willardson 
etal., 1989). Products of alternative splicing of the band 3 protein AE1 gene have been 
characterized in chicken kidney, where it is thought that the variation in transcripts serves 
to determine the membrane domain to which the polypeptides are targeted (Cox et at., 
1995). A similar situation may exist for the various HKa : transcripts; alternative splicing 
may mediate the polarity of expression. In cortical thick ascending limb, an H,K-ATPase 
activity has been described that diminished in rats fed a low K + diet (Younes-Ibrahim et 
at, 1995). Basolateral polarity of expression would be consistent with potassium 
homeostasis in that case. However, no basolateral staining using the anti-HKa 2c antibody 
was seen in this segment of the nephron (discussed below). If the HKa 2b protein is the 
molecule responsible for that activity, its absence in RCCT-28A could be explained by the 
normal expression being in a region of the cortex other than CCD. 

A number of possibilities exist to answer the question of why there are multiple 
H,K-ATPases in the kidney. One possibility is differential regulation. In rat (Kone and 
Higham, 1998) and rabbit (this work) the alternatively spliced HKa : subunit mRNA 
contains multiple upstream open reading frames. This may be associated with an inhibition 
of translation, leading to a decrease in HKa : subunit protein in the cell. Covalent 



79 
modification is another possible means of differential regulation. In this case, 

phosphorylation is an unlikely candidate for differential regulation because although motifs 
recognised by kinases are present, they are not conserved between rat (Kone and Higham, 
1998) and rabbit. Having a different promoter region immediately 5' to the HKa 2c cDNA 
gives rise to potential differences in regulation relative to HKa 2a . Of course, different 
promoter regions giving different responses to conditions such as aldosterone status and 
low K + may account for why there are the three catalytic subunit isoforms HKct,, HKa 2a , 
and HKa 2c . There may also be differences in the kinetics between HKa 2a and HKa 2c . 

Another possibility is localization, both within the kidney and within the cell. Using 
our antibodies, Dr. Jill Verlander and Ms. Robin Moudy conducted immunohistochemistry 
experiments. They found a similar pattern of expression of expression of HKa 2c compared 
to other H,K-ATPase proteins for which localization is known (Wingo et al., 1990; 
Campbell-Thompson et al., 1995; Aim and Kone, 1995; Aim et al., 1996; Haragsim and 
Bastani, 1996). These results imply that the alternative splicing does not confer on the 
protein a different polarity of expression or localization within the kidney. They do imply 
that the HKa 2c protein joins other acid and ion transporters in cell types specialized for 
high transport activity. 

Immunohistochemistry showed that differences in HKot 2c subunit protein cellular 
distribution had no major departures from the distribution of other H,K-ATPase subunits. 
Because the phosphorylation motifs in rat and rabbit alternatively spliced isoforms are not 
conserved, regulation by phosphorylation is a less likely candidate to lead to differential 
regulation. There are myriad possibilities for why there is such a heterogeneity of 



80 
H,K-ATPases in the kidney, such as differential regulation at the gene level by different 

promoter regions, differential regulation of protein synthesis by the short upstream open 

reading frames, or differences in enzyme kinetics. 



H,K-ATPASES IN A RABBIT KIDNEY CORTICAL COLLECTING TUBULE A-TYPE 

INTERCALATED CELL LINE 

Introduction 



The kidney is a well-organized and complicated organ, with many different cell types 
contributing to its morphology and function. Any understanding of the kidney must 
include an appreciation of the processes mediated by each cell type. Expression of 
H,K-ATPase has been shown to be complex in rabbit kidney, so one issue of interest is the 
identification of the cell types possessing the various isoforms of the pump. 
Immunohistochemical evidence was presented in the previous chapter indicating that the 
collecting duct A-type intercalated cell is one of the primary cell types that expresses 
H,K-ATPase catalytic subunit proteins. An independent approach showing that A-type 
intercalated cells express these pumps is to detect H,K-ATPase in a well-characterized cell 
line of intercalated cell origin. A cell line in which H,K-ATPase was present would also 
serve as a model cell system offering the potential for future experiments pertaining to 
renal H,K-ATPases. 

The rabbit cortical collecting tubule cell line RCCT-28A was selected by 
immunodissection (Arend et ai, 1989). Cortical collecting duct was dissected under a 
microscope and the collagenase-dispersed cells incubated in culture dishes to which the 
antibody rct-30 was attached (Spielman et al., 1986). The rct-30 antibody binds 

specifically to collecting duct cells. The resultant cell line was shown to have 

si 



82 
characteristics of the cortical collecting duct A-type intercalated cell (Arend el ai, 

1989;Schwiebert «?/<//., 1992; Dietl eta/., 1989; Bello-Reuss, 1993). The existence of 
H,K-ATPase mRNA, protein, and activity in this cell line was studied to demonstrate that 
this is a cell type that expresses H,K-ATPase. Here we show by RT-PCR that mRNA for 
HKcti, HKa 2a , HKa 2c , and HKP are present in RCCT-28A cells. By western analysis it is 
shown that protein corresponding to the novel alternatively spliced isoform HKa 2c is 
present. Lastly, fluorescence microscopy measurements are used to demonstrate that 
RCCT-28A cells have a mechanism for pH, regulation that is K + -dependent and sensitive 
to the H,K-ATPase inhibitor Sch-28080. Together the data represent very strong evidence 
for H,K-ATPase expression in a cell line derived from the type-A intercalated cell in the 
collecting duct. 

Detection of H.K-ATPase in RCCT-28A Cells 
Detection of H.K-ATPase mRNAs in RCCT-28A cells 

To determine the presence of H,K-ATPase transcripts, we examined whether the 
RCCT-28A cells possessed mRNA for the known H,K-ATPase or a subunits. In the 
initial experiments, there were no H,K-ATPase subunits detectable by northern analysis, 
nor were there RT-PCR products amplified from RCCT-28A cells that could be directly 
visualized by agarose gel electrophoresis. A more sensitive technique, Southern blotting of 
the RT-PCR products using cDNA probes designed to hybridize to the expected products 
was employed to demonstrate the presence of H,K-ATPase subunit mRNA in these cells. 



83 
RT-PCR products were amplified using RCCT-28A cell total RNA as a template. The 

presence of HKp subunit mRNA was observed as a product of approximately 309 bp 

hybridizing to a probe containing nucleotides 304-873 of the rabbit HKP sequence 

obtained by Reuben et al. (1990) in gastric tissues (Figure 5-1 A). Sizes were estimated by 

measurements from the original agarose gel on which a 100 bp ladder was visualized by 

ethidium bromide staining. The "-RT" lanes in this and all subsequent figures depict 

negative controls in which RT was omitted from reactions to show that the RNA template 

was free of contaminants. Products amplified from mRNA isolated from rabbit renal 

cortex tissue were also included in these experiments as a positive control for the RT-PCR 

reaction. The hybridization of a probe specific for HKP to an RT-PCR product of the 

expected size implied the presence of HKP in these cells. 

At the time these experiments were carried out, the existence of alternatively spliced 
HKct: variants had not yet been discovered. Therefore, these experiments were designed 
only to show the presence of HKa : transcript in general, and do not differentiate between 
the HKa 2a and HKa :c species. For HKa : mRNA, a primer pair yielded the anticipated 
product size of 305 bp. This product was hybridized with a probe of nucleotides 
1264-1569 of the HKa 2a sequence, contained in the region now known to be common to 
HKot 2 a and HKa 2c (Figure 5- IB). 

A similar strategy was employed to show that HKa, was present in the RCCT-28A 
cells. Primers were designed to amplify a 61 1 bp region of HKa, mRNA (nucleotides 
2537-3147), and again a product of the expected size was observed to hybridize to a 
probe containing the same 6 1 1 bp region (Figure 5- 1 C). At the time these experiments 



A) 

600 bp 
300 bp 

100 bp 
RT 



C) 



+ 



P 

o 

o 
3 

x 



600 bp 
300 bp 

100 bp 
RT 



B) 



84 




+ 



n 
n 

H 
i 
S3 

oo 

> 




3 

n 

H 

i 
to 

oo 

> 



600 bp 
300 bp 
100 bp 
RT 




+ 



g 

o 

H 
i 
to 

oo 

> 



D) 






800 bp - 






400 bp - 




• 


200 bp - 








W 


71 




— < 


n 




El 


n 






H 




O 

o 

a 

X 


OO 

> 



Figure 5-1 . Southern blots of H,K-ATPase subunit mRNA in RCCT-28A cells. A) 
Products amplified by RT-PCR were shown by Southern blot analysis. PCR primers 
were designed to amplify a 309 bp region within the 3' UTR of H,K-ATPase p subunit. 
B) PCR primers designed to amplify a 305 bp region within the coding sequence of 
H,K-ATPase cc 2 subunit produced the products shown. C) PCR primers designed to 
amplify a 61 1 bp region within the 3" UTR of H,K-ATPase a, subunit produced the 
products shown. D) Restriction digests of the 61 1 bp fragment of H,K-ATPase a,. 
Bam HI digestion was predicted to yield fragments of 423 and 187 bp. Pst I digestion 
was predicted to yield fragments of 353 and 257 bp. 






85 
were ongoing, there were no reports in the literature of HKa, in kidney, so a further step 

was taken to confirm the identity of these products. Restriction digests were carried out 

on these products, and the anticipated size fragments were obtained (Figure 5- ID), further 

evidence that these products corresponded to HKa, mRNA in RCCT-28A cells. 

With further optimization of RT-PCR protocols, including such parameters as 

annealing temperatures and number of amplification cycles, it was found that the RT-PCR 

technique was capable of generating products visible by ethidium bromide staining of 

agarose gels. This was a substantial improvement over the preceding experiments, because 

it offered the opportunity to obtain nucleotide sequences of the PCR products to confirm 

their identity. RT-PCR products were generated and sequenced using RCCT-28A cell 

total RNA as a template. The presence of HK(3 subunit mRNA was observed by 

amplification of a 570 bp cDNA corresponding to nucleotides 304-873 of the sequence 

obtained by Reuben el a/. (1990) in gastric tissues (Figure 5-2A). Nucleotide sequencing 

of the RCCT-28A product confirmed that the amplified product was identical with the 

rabbit HKp subunit mRNA. 

HKa, was also shown to be present in the RCCT-28A cells by the same technique. 
Primers were designed to amplify a 61 1 bp region of HKa, mRNA (nucleotides 
2537-3147), and again a product of the expected size was observed (Figure 5-2B). The 
nucleotide sequence was identical to that reported by Bamberg el a/. (1992) except for 
two single base mismatches. One mismatch was a transition of G->A at nucleotide 2567 
and the other a G^C transversion at nucleotide 3089; neither affected the deduced amino 
acid sequence. 



86 



A) 




B) 




Figure 5-2. HK0 and HKa, subunit mRNA in RCCT-28A cells. PCR products were 
generated using RCCT-28A cell and rabbit renal cortex total RNA. Amplification was 
40 cycles for RCCT-28A cell PCR products and 30 cycles for renal cortex PCR 
products. 100 bp ladder is shown for size reference. 



87 
For HKa 2 mRNA, a primer pair yielded the anticipated product of 305 bp, which was 

identical to nucleotides 1264-1569 of the HKa 2a sequence. This segment was contained in 

the region common to HKot 2a and HKa 2c (Figure 5-3A). To determine whether HKa 2a and 

HKct 2c isoforms were both present in RCCT-28A cells, primer pairs were designed that 

amplify regions specific to each isoform. HKa 2a -specific primers amplify an 86 bp 

(nucleotides 7-93 in the HKoc 2a sequence) RT- PCR product from RCCT-28A cellular 

RNA and nucleotide sequence determination showed the product to be identical to the 

sequence of the HKa 2a cDNA from rabbit cortex (Figure 5-3B). A product of 354 bp 

(nucleotides 24-377 in the HKa 2t sequence) amplified using rabbit renal cortex mRNA as 

template was also generated (Figure 5-3C). The sequence of this product was identical to 

the HKa 2c cDNA sequence from rabbit renal cortex. Therefore, RCCT-28A cells 

contained the mRNAs encoding all three HKa subunits and the HK{3 subunit. Since 

RCCT-28A cells are a clonal cell line, the implication is that individual A-type intercalated 

collecting duct cells may express at least three different H,K-ATPases. 

Detection of HKq subunit protein in RCCT-28A cells 

With convincing evidence of the presence of mRNA for these H,K-ATPase subunits in 
hand, the next logical experiments was to demonstrate that RCCT-28A cells also 
produced H,K-ATPase proteins. We employed the antibodies described in previous 
chapters to determine the H,K-ATPase catalytic subunits present in these cells. Using 
western analysis and one of the antibodies to the common core of the HKoc 2 proteins 
(LLC25 anti-HKa 2 common) and two of the antibodies specific to FIKa 2c (LLC26 and 



A) 



B) 



C) 




88 



Figure 5-3. HKcc 2 subunit mRNA in RCCT-28A cells. PCR products were generated 
using RCCT-28A cell and rabbit renal cortex total RNA. Amplification was 40 cycles 
for RCCT-28A cell PCR products and 30 cycles for renal cortex PCR products. 100 bp 
ladder is shown for size reference. 



89 
LLC27 anti-HKa:,), it was demonstrated that at least the HKa 2c protein was present in 

these cells. The second antibody to the common core protein (LLC24 anti-HKa 2 
common) failed to show reactivity in the RCCT-28A cells in initial experiments, and all 
further western analysis was done using LLC25 (anti-HKa 2 common). Of the two 
HKa 2c -speciflc antibodies, LLC27 (anti-HKa 2c ) gave the least background so it was 
selected for all further western analysis. 

In Figure 5-4A, immunoreactivity of LLC25 (anti-HKa 2 common) to membrane 
proteins of RCCT-28A cells is shown. Reactive protein was detected migrating at an 
approximate molecular mass of 90 kDa, consistent with the presence of HKa 2 subunit 
protein in these cells. Although this broad band may represent a doublet, this has not been 
clearly resolved. Figure 5-4B shows the results of western analysis of RCCT-28A protein 
and the LLC27 (anti-HKa 2c ) antibody. A band was visible corresponding to a molecular 
mass of 90 kDa, consistent with the presence of HKa 2c subunit protein in the RCCT-28A 
cells. Based on these data one may conclude that an HKa 2c subunit protein was present, 
but because two species were not discernable using LLC25 (anti-HKa : common) it was 
not proven that HKa 2a subunit protein was there as well. Given the results using LLC25 
(anti-HKa 2 common) and LLC27 (anti-HKot 2t ) antibodies, it might be argued that only 
HKa 2c was present in RCCT-28A cells. It was only with difficulty that the doublet 
indicative of both HKa 2 proteins were observed in kidney, so not observing a doublet in 
the RCCT-28A cell western should not be taken as proof of the absence of HKa 2a protein. 
Importantly, the preceding mRNA detection experiments showed that both alternatively 
spliced transcripts were present in these cells. 



90 



A) B) 

anti-HKa 2 common anti-HKa 2c 

121 kDa- 

120 kDa- 
78 kDa- 80 kDa- 

50kDa- 
50 kDa - 



P 8 

oo oo 

> > 



Figure 5-4. Western analysis showing presence of HKa 2a and HKa 2c protein in 
RCCT-28A cells. A) Detected with the anti-HKa 2 common antibody LLC25. B) Detected 
with the anti-HKa 2 c-specific antibody LLC27. 



91 
In preliminary experiments involving RCCT-28A cells, the HKa, -specific antibodies 

we produced (LLC22 and LLC23 anti-HKa,) did not show reactivity. However, using 

RCCT-28A cells we provided, Dr. Adam Smolka of the Medical University of South 

Carolina did show reactivity to the HKa, antibody HK12. 18 (personal communication). 

Detection of H.K-ATPase activity in RCCT-28A cells 

H,K-ATPase subunits were not detected in RCCT-28A cells by northern analysis, and 
the subunits were apparently at relatively low abundance by western analysis. These 
results implied that H,K-ATPase expression was at a low level in these tissue culture cells. 
Because mRNA and protein levels were so low, it was necessary to determine a basal 
H,K-ATPase activity in these cells. To confirm by an independent method that the 
RCCT-28A clonal cell line has H,K-ATPase activity as previously reported (Bello-Reuss, 
1993), fluorescence microscopy with digital video image analysis was used to measure pH, 
recovery rates of individual cells after an acid load. 

These experiments were undertaken in the laboratory of Dr. David Weiner, who had a 
decade of experience making similar measurements, and kindly provided his equipment 
and expertise. The development of a technique for measuring H,K-ATPase activity was 
important for future studies undertaken by our lab, and the technique developed here 
offered advantages over those previously used. This technique measures activity of 
individual cells in order to achieve maximal sensitivity and to assess the homogeneity of 
the population of tissue culture cells. Additionally, this technique used the same apparatus 
used to measure H,K-ATPase activity in perfused tubules, allowing comparison to 
activities measured in a system more representative of the kidney in the whole animal. The 



92 
actual protocols used underwent some evolution as described below. Results are first 

described for the early protocols used to provide an appreciation for how the differing 
protocols affected the measurements. Final results obtained by the refined protocol then 
follow. 

The technique used to measure H,K-ATPase activity in this work consisted of 
fluorescence microscopy to observe changes in pH, of single cells while cells were 
perfused by solutions containing various substrates and inhibitors. For example, 
H,K-ATPase activity requires K + entry for intracellular alkalinization. If H,K-ATPase 
activity were the only method of acid extrusion available to an acidified cell, pHi would be 
expected to rise back to its equilibrium pH, in solutions containing K" due to exchange of 
extracellular K + for intracellular FT. In solutions lacking K\ this exchange would be 
absent, and cells could not recover. The rate of pH, recovery (pH units/min) in the 
presence of K + would be proportional to H,K-ATPase activity. Another maneuver that 
was used to identify H,K-ATPase activity was to measure recovery rates in the presence 
and absence of an H,K-ATPase inhibitor. Like the absence of K\ the presence of the 
inhibitor would block recovery of an acidified cell to normal pH,. By inhibition of other 
mechanisms of pH, regulation, H,K-ATPase activity can indeed be isolated and rates 
determined in this way. 

pH, was determined by changes in fluorescence of the pH-sensitive dye BCECF. The 
acetoxymethyl ester of BCECF crosses cell membranes freely, and intracellular esterases 
cleave the ester bond trapping BCECF within the cell. Therefore, BCECF-AM was the 
agent used to load cells with BCECF for pH, measurements. After BCECF loading, cells 
were transferred to the microscope stage. The next step of the experiment was acid 



93 
loading of the cells. This was done using an NR,C1 prepulse. Solution 2 (Table 2-2) 

contained the neutral, membrane-permeant ammonia which entered the cell and 

equilibrated to produce an amount of the weak acid ammonium. Upon removal of 

ammonia from the extracellular solution, intracellular ammonia escaped the cell down its 

electrochemical gradient. Ammonium was trapped within the cell by its membrane 

impermeance, the electronegativity of the cell interior, and the fact that NFL, + is 

transported inwardly by Na,K-ATPase. This trapped ammonium acidified the cells. As 

already noted, following this acidification, measurements may be made of transporters that 

mediate acid extrusion and thus give rise to alkalinization of the cell. Following these 

measurements, the NaVFT exchanger was released from inhibition, and in functional cells a 

robust recovery toward equilibrium pH, for the cell ensues. 

Once pH, for all the cells in the field of view of the microscope was recorded by the 
computer for each time point in the experiment, analysis of the individual cells may begin. 
The FL/1 program allows the selection of single cells, and the pH, is plotted through the 
different changes of solution. Rates of change in pH, for the relevant periods may be 
calculated by a spreadsheet computer program. It is these rates of recovery that were the 
basis of defining H,K-ATPase activity. 

The most robust and ubiquitous mechanism of pH, regulation in cells is the NaTFT 
exchanger. The relatively large contribution to recovery from an acid load mediated by the 
NaTFT exchanger obscured recovery by other mechanisms, so its activity had to be 
blocked for measurements of activities of other H* transporters. In the initial studies of 
this work, Na + removal was used to block NaVFT exchanger activity. In these studies, 
described more fully below, H,K-ATPase activity pH, recovery rates were low. At the 



94 
time, Dr. Weiner was attempting without success to detect H,K-ATPase activity in A-type 

intercalated cells in perfused tubules. A suggestion by a reviewer of a manuscript from Dr. 

Weiner' s laboratory was to utilize the potent amiloride analog EIPA to inhibit the Na7H + 

exchanger instead of using removal of Na\ As seen below, measurements of 

H,K-ATPase-mediated recovery roughly doubled in the presence of Na" with EIPA, and 

this must be regarded as a major improvement of this assay compared to its original form. 

Hays and Alpern (1991) had previously observed an effect of Na + removal on pH, 

regulation by H + pumps, and in its final form, the study of A-type intercalated cells by 

Milton and Weiner (1997) observed a similar effect. 

Another major evolution of these studies was the finding that there were 

time-dependent effects that appear after acid loading. These were never observed in 

control experiments at a time less than 8 min following NH 4 C1 removal. After the 8 min 

period, two separate effects that would disturb rate measurements were seen in some cells. 

The first was that in some cells the recovery would plateau before attaining the normal 

equilibrium, baseline pH,. When Na' was returned to solutions, or when EIPA was 

removed, functional cells fully recovered toward baseline. This plateau effect has been 

observed in previous studies of Na '-independent pH, regulation (Montrose and Murer, 

1986, Montrose etal., 1987). The second confounding effect was that in the continued 

absence of potassium a delayed increase in the pH, recovery rate developed, consistent 

with a delayed stimulation of H -ATPase. These results were consistent with previous 

results by Hays and Alpern ( 1 99 1 ), who described an H -ATPase activity that was delayed 

following an NH 4 C1 prepulse. Bello-Reuss (1993) also detected H -ATPase as well as 

H,K-ATPase activity in RCCT-28A cells. As a consequence, the original protocols calling 



95 
for a period of K + absence followed by a period of K T addition had to be modified. 

Experiments inolving perfused tubules typically involved taking data in absence then 
presence of solution substrates or inhibitors, but in this system there was not sufficient 
time following acid loading for rates to be accurately determined for both K + absence and 
K + presence. Therefore, the final design of the protocol called for making multiple 
separate measurements of K '-dependent recovery rates and IC-independent recovery 
rates. Sch-28080-sensitive recovery rates were also determined in multiple separate 
experiments. With measurements made in this way, there was a statistically significant 
difference between the experimental conditions with the measurements being consistent 
with the presence of an H,K-ATPase in RCCT-28A cells. 

Apical IC-dependent RCCT-28A cell pH, regulation in the absence of Na~. Figure 5-5 
shows a representative tracing of a cell that was acid loaded by an NH 4 C1 prepulse 
{solution 2 added to basolateral chamber) and pH, recovery observed for a period of 5 min 
in the absence of Na\ HC0 3 , or apical K' {solution 3 in apical chamber, solution -I in 
basolateral chamber). Under these conditions, recovery of pH, to physiological range 
mediated by such processes as NaVH exchange, C17HC03- exchange, or apical H7K + 
exchange would be blocked. A population of cells was found in which the mean recovery 
pH; rate was 0.000±0.001 pH units/min under these conditions, not significantly different 
from zero. Upon switching the media perfusing the apical side to solution -/, a 
K + -containing solution, a mean recovery rate of 0.01 5±0. 004 pH units/min was observed, 
consistent with an apically located K -dependent mechanism of pH, recovery. With Na + 
added to the perfusing solution, a robust recovery of pH, toward baseline value was 
observed, presumably mediated by Na'/FT exchange. 



96 



NH4 + 


- 


+ 


- 



Na + 


+ 


- 


+ 



ApK + 



+ 



+ 





5 Minutes 




062096G7 



Figure 5-5. pH f recovery from an acid load by RCCT-28A cells in the absence of Na + . A 
representative tracing of pH f in an individual cell is shown. Periods during which 
solutions contain 20 mM NK.C1, 120 raM Na + , and 5 mM K + on apical (Ap) side are 
indicated.Throughout the experiment 5 mM K + was present on basolateral (Bl) side. Cells 
were acid loaded and recovery was observed in the absence of Ap K + , then in the presence 
of Ap K + . No K + -independent recovery was observed, but in the presence of K + there was 
detectable recovery. Finally, Na + was added back and robust recovery was observed. 



97 
K + -dependent RCCT-28A cell pH, regulation in the presence of Na'. Because of the 
rather modest level of H,K-ATPase activity detected in these cells, and because of results 
obtained by Milton and Weiner (1997) indicating that Na* must be present for optimal 
measurements of H,K-ATPase activity by this technique, we undertook studies in which 
Na + was present during the pH, recovery period following an acid load. The cells were 
acid loaded by symmetrical addition of solution 5 containing 1 uM EIPA. In the absence 
of K + (symmetrical solution 6), a recovery rate that was not significantly different from 
zero (0.00 1±0. 003 pH units/min) could be measured in three separate cell preparations. A 
K + -dependent recovery (symmetrical solution J plus 1 uM EIPA) could be observed 
(0.032±0.006 pH units/min, P< 0001 vs K + -free perfusion). There was approximately a 
three-fold activation of K'- dependent recovery rate pH, attributable to the presence of 
Na + in the perfusing solutions. 

Independent, EIPA-sensitive RCCT-28A cell pH, regulation. The following 
measurements represent the conclusive experiments, in which Na + is present for maximal 
H,K-ATPase activity and measurements were made during periods in which there were no 
K + -independent artifacts. A representative tracing of a cell acid loaded by an NR.C1 
prepulse (symmetrical solution 5 plus 1 uM EIPA) and pH, recovery observed is shown in 
Figure 5-6A. In a majority of RCCT-28A cells there was significant EIPA-insensitive pH, 
recovery in K + -containing solutions (symmetrical solution 1 plus 1 uM EIPA). For 136 
cells from six independent passages of RCCT-28A cells, the mean recovery rate was 
0.022±0.005 pH units/min. In the absence of K' (symmetrical solution 6 plus 1 uM 
EIPA) during the period following acid load no immediate pH, recovery was observed 
(Figure 5-6B). In five separate experiments (129 cells), the K' -independent pH, recovery 



NIL 



98 



+ 



EIPA - 




+ 



A) 



X 
D, 



5mMK* 




B) 



OmMK> 




c) 



lOuMSCH 




Figure 5-6. pHi recovery from an acid load by RCCT-28A cells in the presence of 
EIP A. Representative tracings of pH, in individual cells are shown. Periods during which 
solutions contain 20 mM NH 4 C1 and 1 mM EIPA are indicated. Cells were acid loaded 
and recovery was observed in the presence of K + (A), in the absence of K + (B) and in the 
presence of Sch-28080 (C). 



•■ 






99 
rate averaged 0.004±0.002 pH units/min (P<0.01 vs. 5 mM K + ). The K + -independent, 
EIPA-sensitive pH, recovery rate measured immediately after acid loading was less than 
that measured during the protocols in which measurements were delayed with respect to 
acid loading. 

Sch-28080- and EIPA-sensitive RCCT-28A cell pH, regulation. To test the 
hypothesis that K + - dependent pH, recovery from an acid load resulted from H,K-ATPase, 
the effect of the classical H,K-ATPase inhibitor Sch-28080 (10 uM) was examined on 
RCCT-28A cell pH, regulation (Figure 5-6C). After acid loading in solution 7 containing 
Sch-28080 plus E1PA, cells were perfused in a K' -containing solution (solution 6 with 
Sch-28080 plus EIPA). Using this protocol, 109 cells from four separate cell preparations 
had a mean recovery rate of 0.002±0.002 pH units/min (P<0.05 vs. 5 mM K\ P=NS vs. 
mM K + ) in the presence of 1 uM Sch-28080. 

Buffer capacity and pH, after acid loading. The effects of the various protocols 
performed in these experiments might be explained by differences in buffer capacity or the 
degree to which cells are acidified by the NH4CI pre-pulse. Although these experiments 
directly measured the rate of pH, change, the variable of interest is proton flux; these two 
values are directly related by buffer capacity. Thus, buffer capacity had to be considered to 
avoid misconceptions concerning proton tlux changes based on observations of pH, 
recovery rates. Buffer capacities measured by the NH3/NH4* technique (Boyarsky et al, 
1988) in the presence of K\ absence of K', and presence of Sch-28080 were 15±1 mEq 
H7pH, U/L cell volume, 16±1 (P=NS vs. 5 mM K + ), and 16±2 (P=NS vs. 5 mMK + ), 
respectively. Therefore, changes in pH, recovery rates among the protocols could not be 
attributed to changes in buffer capacities. 






100 
Some mechanisms of pH, recovery have recovery rates that are influenced by absolute 

pHi, with a tendency for greater acidification to stimulate higher rates of pH, recovery. 

Therefore, a change in the rate of recovery might have been due to differences in absolute 

pHi after acid loading. Actual nadir pH, observed in these experiments in the case of K + 

presence, K + absence, and Sch-28080 presence measured 6.64±0.06, 6.59±0.08 (P=NS vs. 

5 mMK + ), and 6.54±0.03 (P=NS vs. 5 mM K + ). Consequently, changes in pH, recovery 

rates among the protocols was not due to the nadir pH,. 

Discussion 

RCCT-28A cells contained the mRNAs encoding all three HKa subunits and the HK0 
subunit. Since RCCT-28A cells are a clonal cell line, the implication is that individual 
A-type intercalated collecting duct cells may express at least three different H,K-ATPases. 
The HKa subunit mRNA detected in RCCT-28A cells gives rise to at least the HKa, and 
HKoc 2c proteins. It is of interest that the most novel subunit, the alternatively spliced HKa 2c 
protein is indeed present in the cortical collecting tubule A-type intercalated cell line, in 
agreement with the expectations brought by the immunohistochemistry studies. And like 
the immunohistochemical evidence both with LLC22 (anti-HKa,) and in previous work by 
others, the experiments with RCCT-28A cells adds to the evidence that HKa, is also a 
transporter expressed in the collecting duct A-type intercalated cell. 

We elected not to pursue detection of HKp, the putative partner to HKa,, due to time 
constraints and because of our interest in characterizing our own antibodies. It has quite 
recently (less than two months ago) been reported that NaKp, is a likely partner to HKa 2c 



101 
(Dubose etal, 1998; et al Kraut et al., 1998), so detection of that subunit has also not 

been done. The ubiquity of NaKp, in the kidney and in the whole animal would make a 

positive result by western analysis very difficult to interpret in terms of relevance to 

H,K-ATPase 

We have provided independent confirmation of Bello-Reuss' (Bello-Reuss, 1993) 
report that RCCT-28A cells have H,K-ATPase activity. Either the presence of the 
H,K-ATPase inhibitor Sch-28080 or the absence of K f virtually abolished the intracellular 
alkalinization rates of RCCT-28A cells following an acid loading (Figure 5-7). When K + 
was present on only the basolateral side of RCCT-28A cells, mean recovery rates were not 
significantly different from zero. In the presence of K', RCCT-28A cells possessed a 
mechanism for pH, recovery that exceeded the rates observed without K + or with 
Sch-28080. These results constituted an independent confirmation of apical H,K- ATPase 
activity in RCCT-28A cells. 

Studies in heterologous expression systems (Codina et al, 1996; Cougnon et al, 
1996; Lee et al, 1995; Modyanov et al, 1995) and in a mammalian expression system 
(Grishin etal, 1996) suggested that an H,K-ATPase with the HKa ; subunit was less 
sensitive to Sch-28080 inhibition than the HKai pump, thus the differential sensitivity of 
the two enzymes offered the possibility of discriminating between the two at the level of 
activity. In this study the IC-independent and Sch-28080-sensitive pH, recoveries were 
similar, arguing that HKa, is the isoform responsible for basal levels of H,K- ATPase 
activity in RCCT-28A cells. Buffin-Meyer et al. (1997) observed a like situation in rat 



102 



E 



5. 



33 



0.030 



0.025 



0.020 



0.015 



0.010 



0.005 -- 



0.000 





































* 
_^ ** 












■■ 



5mMK + 



0mMK + 



lOuMSCH 



Figure 5-7. Summary of the rates of pH; recovery from an acid load by RCCT-28A 
cells. Mean rates of recovery are indicated by unfilled (presence of K + ), black (absence 
of K + ), and stippled (in the presence of Sch-28080 with K + present) bars. * P<.01 vs. 5 
mM K + . ** P<.05 vs. 5 mM K* P=NS vs. K + . 



103 
CCD, in which H,K-ATPase activity was abolished by Sch-28080 under normal 

conditions, and resistant to Sch-28080 only during K* depletion, suggesting up-regulation 

or mobilization of HKa 2 pumps. If basal H,K-ATPase activity in RCCT-28A cells is 

mediated primarily by HKa,, then post-transcriptional regulation may explain the 

functional silence of the HKa: enzymes. However, there is not yet a clear understanding 

of the Sch-28080 inhibitor profile. In the mammalian expression system (Grishin etai, 

1996), the HKa : activity is inhibited by Sch-28080 at lower concentrations than in the 

heterologous expression systems. Also, it was not certain that the P isoform paired with 

the HKa : subunit in the expression systems is the one that associates with it in vivo. 

Sch-28080 may have an artificially reduced potency in expression systems due to the lack 

of association of the correct P subunit or some other indirect effect, therefore conclusions 

reached based on the inhibitor profile must be interpreted cautiously. 

HKct 2 is sensitive to the inhibitor ouabain (Modyanov el al., 1995; Codina et al. 1996; 

Cougnon et al, 1996; Grishin et al, 1996), while HKa, has no apparent sensitivity, 

suggesting a potential means to discriminate on a pharmacological basis between the 

activity of the two isoforms. However, the primary action of ouabain is inhibition of 

Na,K-ATPase, so application of ouabain would disturb the Na* and K 4 equilibria of the 

cell. Indeed, in preliminary experiments discussed above, H,K-ATPase activity was 

observed to be profoundly affected by the presence or absence of Na'. Therefore, any 

effect of ouabain on apparent activities of the a : subunit H,K-ATPases could not be 

distinguished from a secondary effect due to inhibition of Na,K-ATPase. Only when a 



104 
great deal more is known about the effect of specific inhibitors on H,K-ATPase isoforms, 

including any species differences in their potency, would inhibitor studies be valuable. 

Fluorescent measurements of pH, recovery rates attributable to H,K-ATPase activity 
in microperfused CCD in previous studies (Constantinescu et al., 1997; Silver et al., 1993; 
Silver et al, 1996; Weiner and Milton, 1996, Milton and Weiner, 1997; Silver et al, 
1997) have yielded values between 0.03±0.01 and 0. 129±0.045 pH U/min. These values 
are greater than those in the present study, but transport activity in continuous cell lines is 
typically less robust than that of microperfused tubules. Another continuous cell line of 
renal origin, a mouse inner medullary collecting duct cell line, has been studied using these 
techniques and recovery rates of 0.055 ±0.009 were observed Ono et al, (1996). Species 
difference and axial heterogeneity of the collecting duct may explain the difference in rates 
between the two renal cell lines. 

A major goal of this project was to define which cell type or types in the kidney 
contain H,K-ATPase. The R.CCT-28A cell line, which has many characteristics of the 
collecting duct acid-secreting intercalated cell, clearly expresses several H,K-ATPases. 
Although mRNA and protein level were low, there was detectable H,K-ATPase activity in 
these cells. Multiple H,K-ATPase isoforms may contribute to this activity. 

Understanding of FT and K + transport in the collecting duct by H,K-ATPases must 
involve investigations into the kinetics and regulation of each isoform. Each H,K-ATPase 
isoform might have been limited to a different single cell type rather than a single cell 
containing multiple H,K-ATPases. It was important to determine which of these cases 
actually occurs. An interesting aspect to these studies is the implication that regulatory 
processes of the various H,K-ATPase isoforms reside in a single cell type. This would 



105 
allow an interplay at the intracellular level between isofonns, perhaps by crosstalk between 

signal transduction pathways, or perhaps even by physical interaction between different 

H,K-ATPase subunit isofonns. 



PERSPECTIVE AND FUTURE DIRECTIONS 
Multiplicity of HK-ATPase Isoforms in the Kidney 

Just in the last decade it has become known that H,K-ATPase is one of the enzymes 
that contributes to regulation of acid/base balance and potassium homeostasis by the 
kidney (Doucet and Marsy, 1987; Wingo, 1989). Only recently have the molecules that 
mediate this activity in the kidney been identified (Modyanov el al., 1991; Callaghan etal, 
1995; Ahn and Kone, 1995; Kone and Higham, 1998). This year, evidence was provided 
by two independent investigations that the NaK(3, subunit also is a participant in 
H,K-ATPase activity (DuBose el al., 1998; Kraut el al., 1998). These studies showed that 
NaKp, subunit was the long-sought p subunit that associates with the H,K-ATPase 
catalytic subunit discovered in distal colon by Crowson and Shull (1992). Thus, there is a 
great deal of complexity at the molecular level behind the H,K-ATPase activity that was 
observed in the kidney. 

The studies described in this dissertation address the molecular nature of 
H,K-ATPases in the kidney. A novel HK[3 subunit variant mRNA (HKp') specific to renal 
medulla was found by northern analysis. In addition to the canonical transcript, the novel 
mRNA was expressed in the renal medulla, but not in renal cortex or stomach. The HKp' 
subunit mRNA may have been a product of medulla-specific alternative splicing at the 5' 
end of the HKP gene. There was individual variation in rabbits with respect to 

106 



107 
relative abundance of HKp and HKp' subunit mRNA in medulla of the kidney, but HKp' 

was apparently not regulated by dietary K restriction. The existence of HKp' shows that 

the nature of H,K-ATPase p subunits, as well as a subunits, is complex and must be 

considered in understanding H,K-ATPase function in the kidney. 

A number of H,K-ATPase a subunit cDNAs have been cloned and sequenced from 

various species. They might be initially characterized by their tissue distribution. First, 

there was the well-known gastric isoform HKot, subunit cloned from rat (Shull and 

Lingrel, 1986), pig (Maeda el al., 1988), human (Maeda el al., 1990), rabbit (Bamberg et 

al, 1992), frog (Mathews el al, 1995), and mouse (Mathews et al, 1995). Second, there 

were H,K-ATPase catalytic isoforms cloned from human axillary skin (Modyanov et al, 

1991), rat distal colon (Crowson and Shull, 1992), guinea pig distal colon (Watanabe et 

al, 1993), and a partial cDNA from rabbit kidney (Fejes-Toth et al. 1995). These were 

not detected in stomach, but were expressed in numerous other tissues, primarily distal 

colon. Thus far, at most two P-type H,K-ATPase a subunit isoforms have been identified 

in any one species. Alternatively spliced variants of the non-gastric isoforms have been 

found in rat (Kone and Higham, 1998) and rabbit (Campbell el a/., 1998). 

An H,K-ATPase catalytic subunit has been cloned from the bladder of the toad Bufo 

marimis that had a tissue distribution that contrasted with that seen in mammals (Jaisser et 

al, 1993). While northern analysis detected the toad HKa transcripts in bladder, it was 

not observed in either stomach or distal colon. This may reflect differences in physiology 

between the toad and mammalian species. For example, the toad may not require distal 



108 
colonic K + reabsorption for K + homeostasis. On the other hand, the toad HKa may be the 

first evidence of an H,K-ATPase subunit that has escaped detection in mammals. 
Controversy persists over the nomenclature of H,K-ATPase subunits. Some 
investigators classify the rat non-gastric HKa as a different H,K-ATPase than the human 
non-gastric HKa. This is based on qualitative differences in inhibitor profile observed for 
rat and human non-gastric isoforms in heterologous expression systems. Under their 
nomenclature, rat and human H,K-ATPase subunits are referred to as HKoc 2 and HKa*, 
respectively. Quantitative differences in sensitivities to inhibitors may not be a valid 
measure by which to classify orthologous groups. The Na,K-ATPases also have widely 
differing qualitative differences between species in their response to inhibitors. As an 
example ouabain, the widely used "specific" inhibitor of Na,K-ATPases, is highly effective 
against the pumps in most species other than rat. For rat NaKa,, ouabain is effective only 
at higher concentrations, but the rat NaKa, is not thought of as a different NaKa, than 
other species. To classify H,K-ATPase isoforms, it might be more valuable to first look at 
tissue distribution. This would lead to three classes of H,K-ATPase catalytic subunits. 
HKa, would continue to denote the gastric H,K-ATPases catalytic subunits, HKa 2 would 
refer to the non-gastric isoforms of H,K-ATPase a subunits, and HKa 3 would denote the 
toad bladder isoform. It became one of the goals of this dissertation to determine how the 
rabbit HKa isoform related to other H,K-ATPases. Rabbit, rat, and human non-gastric 
H,K-ATPase cDNA sequences will best fit into a single group based on their tissue 
distribution and on their phylogenetic relationship. 



109 
A search for novel P-type H,K-ATPases expressed in the kidney was performed. The 

purpose of this was twofold. First, a rabbit HKa? sequence was needed to design reagents 

for study of H,K-ATPase regulation and function in the kidney. In addition, the sequence 

would be useful to compare and contrast with HKa 2 sequences found in other species to 

examine their relationship and find important conserved motifs. Second, it was hoped that 

such a search would find any previously unknown H,K-ATPase catalytic subunit isoforms. 

The search as conducted was by no means exhaustive, once an interesting fragment of 

sequence was found, no further attempts were made to find others. However, other 

investigators also looking for novel H,K-ATPases in rabbit kidney also found the same 

isoform as described in this work (Fejes-Toth el al., 1995; Jaisser, personal 

communication). Because of the numerous attempts to expand the number of 

H,K-ATPases known in human and rat (Shull and Lingrel, 1987; Sverdlov el al, 1987; 

Modyanov et al, 1991; Crowson and Shull, 1992), it must now be regarded as less likely 

that completely novel mammalian isoforms will be found. 

An HKa subunit isoform was found that had high homology to human and rat HKa 2 . 

The relationship of the rabbit HKa subunit isoform to other H,K-ATPases and 

Na,K-ATPases was computationally analyzed by the distance and maximum parsimony 

algorithms. It was found that sequences for rat and human HKa : subunit isoforms were 

more closely related than HKa, or the Na,K-ATPases. The HKa 3 subunit sequence of 

Bnfo marinus was slightly more homologous to the HKa : subunit sequences than to the 

HKa, sequences. Without more H,K-ATPase sequences related to HKa 3 , its exact 

relationship to other H,K-ATPases will be difficult to judge. 






110 
Kone and Higham (1998) had found an alternatively spliced 5' end of an mRNA that 

they designated HKa 2 b. In the work described here, there were two 5' ends found in 
rabbit as well. The translation start sites of the rabbit and rat alternatively spliced variants 
are not conserved. In the rat HKcc 2b mRNA, the start codon is downstream of the splice 
site (Kone and Higham, 1998), and in the rabbit HKa 2c the start codon is upstream of the 
splice site. For this reason, the rabbit H,K-ATPase catalytic subunit that is the product of 
alternative splicing was designated HKcc 2c . However, the pattern of exons and introns at 
the 5' end of the HKa 2 gene in rat and rabbit was determined to be the same. The 
relationship shown by phlogenetic analysis and the similarity of alternative splicing implied 
that rat and rabbit HKa 2 do indeed represent the same gene in different species. An 
alternatively spliced transcript has not so far been observed for human HKa 2 , but that does 
not constitute absolute evidence that a similar variant was not transcriptionally competent 
in human. The human HKct 2 gene sequence (Sverdlov et a/., 1996) predicts a translational 
start codon that was different from both rat and rabbit, downstream of the splice site, but 
upstream of the first in-frame ATG known for rat. Based on phylogeny, the human HKa 2 
was also an ortholog, with its alternatively spliced transcript thus far unseen. 

Because of the lack of conservation of splice sites between rat and rabbit alternatively 
spliced HKa 2 , it was necessary to determine that the start codon in rabbit that gave rise to 
the longest open reading frame was indeed used. Western analyses using the anti-HKa 2 
common and anti-HKa 2c antibodies showed a pattern of reactivity consistent with 
theHKa 2c protein being larger than theHKa 2 , protein. Therefore, the predicted start codon 
is the one used in rabbit. 



Ill 

The work involved in this dissertation did not succeed in answering the question of 
why so many H,K-ATPase isoforms exist. With respect to HKa :c , regulation by covalent 
modification is rendered a less attractive possibility by the lack of conservation of the 
amino terminal end. The potential phosphorylation sites that were identified by computer 
analysis in the rabbit are within the amino terminal region that is missing in the rat. 
Immunohistochemistry showed that the localization of HKa 2c within the kidney did not 
differ widely from that previously found for HKa,. HKa 2c is apparently not the molecule 
that is responsible for the basolateral H,K-ATPase activity characterized in proximal 
tubule and thick ascending limb of Henle by Younes-Ibrahim et a/. (1995). 

The existence of an alternatively spliced isoform offers the possibility of more complex 
transcriptional regulation. One alternatively spliced isoform may be responsive to different 
stimuli than another. Perhaps the protein arising from the alternative splicing has different 
properties, such as kinetic parameters, degradation rates, or internalization rates. Protein 
interactions have been seen to have major effects on such catalytic properties as ATP 
binding affinity (Morii et ai, 1996), and the amino terminal extension could easily play a 
role in intra-molecular interaction with other H,K-ATPase molecules or with other 
proteins. Regulatory proteins could bind the extension to activate or inactivate the 
enzyme. There is also a possible role of the upstream open reading frames in reducing the 
efficiency of translation. All these possibilities deserve attention, and work is under way in 
the Cain laboratory to explore the ways in which HKa* and HKa :c differ functionally in 
an expression system. 



112 
Cell Type Specificity of H.K-ATPase in the Kidney 

H,K-ATPase activity is found at highest level in collecting duct, with some 
investigators reporting activity in thick ascending limb (Younes-lbrahim et a/., 1995). In 
situ hybridization studies have been conducted that show the distribution of HKcti (Ahn 
and Kone, 1995) and HKp (Campbell-Thompson et a/., 1995). Both HKa, and HKp 
share a similar distribution, primarily in collecting duct intercalated cells. A similar cellular 
distribution was obtained by immunohistochemistry for HKa,, with apical polar 
distribution (Wingo et a/., 1990; Haragsim and Bastani, 1996). In immunohistochemical 
studies using the anti-HKa 2 c carried out by Dr. Jill Verlander, a similar localization of 
immunoreactivity was seen, indicating that HKa :c was expressed in generally the same cell 
types and polarity as HKa, and HKp. The implication was that HKa 2c represents an 
H,K-ATPase that has the same qualitative function as other H,K-ATPases, but may have 
some regulatory differences. The observation that HKa,, HKa 2a , HKa 2c , and HKp were all 
detected in an acid-secreting intercalated cell line of the cortical collecting duct adds to the 
weight of the evidence that one of the cell types in vivo expressing H,K-ATPase subunits 
were A-type intercalated cells. It was highly interesting that all these acid pumps, like the 
V-type H-ATPase (Haragsim and Bastani, 1996) are expressed in the same cells. These 
cells are uniquely differentiated to mediate acid secretion. Intercalated cells undergo 
hypertrophy and hyperplasia in animals facing an acid compromise. Because of the 
capability of that single cell type to increase the overall capability of the kidney for 
handling an acid state, it is logical that the molecules that participate is acid extrusion may 
be found in that cell type. 



113 
Future Studies 

The studies detailed in this dissertation naturally lead to many questions and must 
leave many of these questions unanswered. The most pressing question upon finding a 
novel enzyme or variation on an enzyme is what is the significance of the novelty? In the 
cases of HKP' and HKa 2c , efforts were begun to answer this question, but the answer lies 
elsewhere in experiments yet to be done. Dietary K + restriction was tested as a possible 
regulatory factor, and mineralocorticoid levels and acid/base status are likely candidates 
that remain to be examined, lmmunohistochemistry failed to reveal any large departure of 
HKa 2c localization compared to other H,K-ATPases in the kidney, but this localization 
could change upon disturbances in acid/base, K, or mineralocorticoid status. There are no 
immunohistochemical studies available for any of the H,K-ATPases under these three 
conditions. 

One would expect that if there were a third mammalian H,K-ATPase a subunit, with 
the number of investigators searching, that it would have been found. Perhaps there is 
some sequence-specific difficulty in its amplification or cloning. A difference in size or 
secondary RNA structure could affect the efficiency with which a novel sequence was 
amplified. Perhaps RT-PCR reactions using degenerate primers ought to be attempted in 
greater numbers to increase the chance of finding a truly novel H,K-ATPase, one that is 
either difficult to process or at a much lower level than other P-type ATPases. The 
experiment would have to be done many more times. This is practical given current prices 
for reagents and sequencing. The HKa 2 subunit mRNA fragment was found in the third 



114 
reaction; if a novel sequence were present at tenfold less copies per reaction or ten-fold 

lower efficiency, it should be found in thirty reactions. 

There is evidence of an H,K-ATPase in thick ascending limb having different 
properties than known H,K-ATPases (Younes-lbrahim el al, 1 995; Buffin-Meyer et al., 
1997). This is tantalizing evidence that more H,K-ATPase molecules may exist. RT-PCR 
is now being carried out in glass capillaries with vanishingly small amounts of starting 
material. Perhaps this could help by allowing individual regions of nephron and collecting 
duct to be tried as sources for novel H,K-ATPases. 

To look at the question of novel H,K-ATPases a different way, it will over the next 
five to ten years be a simpler task to put an upper limit on how many P-type H,K-ATPases 
there are. If this number were raised, it would lend weight to the argument for more 
attempts at finding novel H,K-ATPase molecules. All of the P-type H,K-ATPases pointed 
to by earlier work by Shull and Lingrel ( 1 987) and Sverdlov el al. ( 1 987) have now been 
associated with a specific enzyme. Southern analysis of restriction digested genomic DNA 
probed at low stringency by probes corresponding to well-conserved regions of P-type 
ATPases could help determine the number of P-type Na,K- and H,K-ATPases exist. With 
the sequencing of the human genome, such searches will be able to be carried out in silico. 
This will allow many more parameters in the experiment. 

Alternative splicing has now been observed in rat and rabbit HKa : . It would be quite 
interesting to try to observe alternative splicing of a transcript in human HKa 2 as well. A 
positive result would constitute one further piece of evidence that the rat, rabbit, and 
human genes are analogs of the same gene in different species. Another piece of 
interesting evidence could come from chromosomal location of these genes. If the genes 



115 
were found in syntenic regions, it would strengthen the argument for their being analogs 

of the same gene in different species considerably. 

Much work remains to be done in appreciating the localization of the H,K-ATPases in 
the kidney. Double-labeling immunohistochemistry using well-characterized markers 
would help define the cell types in which each H,K-ATPase subunit isoform was 
expressed. Quality antibodies are available for carbonic anhydrase, a marker for 
intercalated cells, and for band 3 protein, a marker for A-type intercalated cells. This 
would help refine our knowledge of the cell types in which H,K-ATPase is expressed. At 
present, an experienced eye is the main tool for determining cell types. With 
double-labelling experiments, the conclusions would be more concrete. 

A major advance in making H,K-ATPase activity measurements would be to find 
inhibitors that worked at low concentrations with high specificity. Characterization has 
been limited to the effects of Sch-28080 and ouabain action on HKot : , but literature is 
conflicting with respect to the concentrations observed for inhibition constants (Modyanov 
etal., 1995;Codinat?/al., 1996; Grishin el al., 1996; Cougnon el al., 1996). These 
conflicts need to be resolved, and other H,K-ATPase inhibitors, such as omeprazole or 
A80915A, might be tried to measure their action on the HKa 2 species. These experiments 
would preferably be conducted in mammalian expression systems. Members of the Cain 
laboratory are in the process of developing such a system. With useful information about 
inhibitor profiles, studies in perfused tubules might be performed to determine the cell 
types that have the various H,K-ATPase activities. Protocols for determining cell types 
functionally in perfused tubules are well known. 



116 

To answer broader questions, such as how does the kidney respond to acid/base 

disturbances, K r restriction, or mineralocorticoid levels, new techniques will be useful. 
There are now blot arrays available that have many cDNAs attached. These may be 
probed with labelled mRNA from controlled and treated animals and differentials in many 
mRNA levels observed in parallel. Each cDNA had an assigned location on the blot, so 
one can quickly find what transcript is being regulated. For a more all-encompassing 
approach to these experiments, microarrays may be used that can have even more cDNAs 
on them. These methods study many regulatory processes in parallel, and could be a quick 
way to pinpoint interesting responses. However, these techniques require first that the 
responsive element was identified and sequenced. That was the goal of this work, to 
identify and characterize H,K-ATPase molecules, elements that play a role in the 
biological processes carried out in the kidney. 

To that end, variants of HKPand HKct 2 have been found and their sequences 
determined. Such tools as cDNA probes and antibodies have been produced. These are a 
prerequisite for determining the significance of the novel H,K-ATPases. It was shown that 
HKa 2c protein is expressed and that the predicted start codon is used. Evidence by 
immunohistochemistry and the presence of mRNA and protein in an A-type intercalated 
collecting duct cell line showed that acid-secreting intercalated cells are a major cell type 
in which HKa 2c pumps may be found. The HKct 2c pump adds to the complement of 
transporters that may mediate acid secretion and K' reabsorption in those cells. These 
results define H,K-ATPase varieties that were not known when this work began. 



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BIOGRAPHICAL SKETCH 

Grady Campbell was born in Clearwater, Florida, on February 1, 1955. He graduated 
from Charles E. Jordan High School of Durham, North Carolina, in June 1973. The 
following September he entered Emory University in Atlanta, Georgia, and in June 1977 
he received a Bachelor of Science degree, with a major in physics. In August of that year 
he began employment at EG&G ORTEC in Oak Ridge, Tennessee. In the fall of 1979 he 
enrolled as a part-time student at the University of Tennessee, Knoxville, and began study 
toward a master's degree under the direction of Carrol R. Bingham, with a major in 
physics. His thesis was entitled The Decay of Mass-separated l93 Pb to I93 T1; the degree was 
awarded in August 1985. In April of the following year he began employment at California 
Institute of Technology in Pasadena, California In August 1992 he enrolled as a graduate 
student in the Department of Biochemistry and Molecular Biology at the University of 
Florida in Gainesville, Florida. That December, he joined Brian D. Cain's laboratory where 
he carried out the work described in this dissertation. Upon completing the requirements 
for his degree, he is joining Frank W. Booth's laboratory at the University of 
Texas-Houston Medical School in Houston, Texas, where he will find and study genes 
involved in conferring the benefit of exercise on cardiovascular disease. 



129 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 







Vt&** — <-/ C£t*~ 



Brian D. Cain, Chair 
Associate Professor of Biochemistry 
and Molecular Biology 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 

'''Susan C. Frost 
Associate Professor of Biochemistry 
and Molecular Biology 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 




licnael S. Kilberg 
Professor of Biochemistry and 
Molecular Biology 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 




Harry S. Nic 

Professor of-£iochemistry and 
Molecular Biology 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality 
dissertation for the degree of Doctor of Philosophy. 




Charles S. Wingo 
Professor of Physiology and 
Pharmacology 



This disssertation was submitted to the Graduate Faculty of the College of Medicine 
and to the Graduate School and was accepted as partial fulfillment of the requirements for 
the degree of Doctor of Philosophy. 

August 1998 




Dean, College of Medicine 
Dean, Graduate School